UNIVERSITY OF GHANA THE 3&LIviE~ LIBRARY ' BAX m LIBRARY THESES 1 . Balme Library theses are available for consultation in the Library. They are not normally available for loan, and they are never lent to individuals. 2. All v/ho consult a thesis must sign an undertaking not to copy or quote from it Tdthout the consent of the author and of this University. University of Ghana http://ugspace.ug.edu.gh STUDIES ON SURVIVAL OF TRACHYSPHAERA FRUCTIGENA Tabor & Bunting CAUSING MEALYPOD DISEASE OF COCOA THEOBROMA CACAO L. A Thesis presented by FHINEAS MARAMBA, B.Sc . (Hons.) in fulfilment of the requirement for the M.Sc. Degree of the University of Ghana JUNE, 1973. From: The Department of Botany, University of Ghana, LEGON. University of Ghana http://ugspace.ug.edu.gh Qt 77 684- University of Ghana http://ugspace.ug.edu.gh I, the undersigned, PHINEAS MARAMBA, author of this thesis, do hereby declare that the work presented in this thesis "STUDIES ON SURVIVAL OP TRACHYSFHAERA FRUCTIGENA Tabor & Bunting CAUSING MEALYPOD DISEASE OF COCOA THEOBROMA CACAO L" was done entirely by me in the Department of Botany, University of Ghana, Legon, fr*m July, 1972 to August, 1973. This work has never been presented either in whole or in part for any other degree of this University or elsewhere. Phineas Maramba, B.Sc. (Hons.) Botany (Legon) University of Ghana, LEGON. University of Ghana http://ugspace.ug.edu.gh ABSTRACT Conidia of T. fructigena did not germinate at any relative humidity from zero to 10C% R.H. Germination in distilled water^ was very poor, less than 2.0 percent at the optimum temperature, 25 C. PDA encouraged 96.8 percentage germination at 25°C. The oonidia germinated very poorly on PDA at 35°C and a temperature of 40 C killed the conidia in approximately 1 2 hours. T, fruotigena conidia would only survive in atmospheres of extremely high humidity. Best survival occurred at 10($ R.H. and 90.0, 2 9 . 7 and 0 .6 percent of the conidia were viable after 1 0 , 20 and 40 days' storage, respectively. In comparison more than 50 percent of the spores perished in 20 minutes at 9Q& R.H. The conidia were well conserved in water and they survived longer than at 10Q$ R.H. Conidia at humidities from zero to 10Q/o R.H. eventually collapsed. Conidia exposed to atmospheres of humidities below 75^ > R.H. shrank instantly. There was increasing delay of shrinkage with rise in rela­ tive humidity above 75^ R.H. Total shrinkage was observed after 1 hour, 1 § hours, 24 hours, 1 5 days and 30 days, respectively in conidia stored at 80, 85, 90, 95 and 10C$ R.H. The conidia were preserved longer at 20° than 28°C. Light up to 467.5 lux intensity had no effect on stored conidia. Removal of water from the spore by plasmolysis in 0.7M potassium nitrate, sodium chloride and sucrose solutions was not as damaging as loss of water during desiccation, and between 79*5 and 96.0 percent of the conidia endured an hour's plasmolysis. Damage only set in after prolonged immersion in the plasmolysing fluid beyond 1 2 - 2 4 hours. No germination occurred in either non-sterile or autoclaved extracts of soil and partially decomposed leaves from coooa farm. The fungistatic principle was seemingly non-volatile and non-fungicidal Highest survival of more than 50 percent was obtained in extracts of pH 8.0. Conidia, both live and dead, buried in soil on slides were attacked by other microorganisms. Conidia killed before introduction into the soil, however, suffered considerably greater attack and were lysed very quickly. T- fructigena showed limited competitive saprophytic ability. Reasonable percentage of baits was colonized only when large quantities of inoculum were used or when T. fructigena had earlier access to the bait than the other soil microorganisms. Sodium nitrate alone or a combination of sodium nitrate and dextrose encouraged greater sapro­ phytic survival than ammonium tartrate, peptone or glucose applied separately. University of Ghana http://ugspace.ug.edu.gh - 4 . - CONTENTS I INTRODUCTION II LITERATURE REVIEW III MATERIAL AND GENERAL METHODS (i) Material ... (ii) General Methods (a) Spore Germinating Tests 1„ Slide Method 2„ Agar Plate Method (b) Assessment of Conidiura Germination (c) Humidity Chambers ... (d) Maintenance of Constant Relative Humidity (e) Spore Prints for Longevity tests (f) Maintenance of Stock Culture (g) Production of Conidia (h) Incubation (i) Aqueous Extract of Soil (j) Methods of Sterilization (k) Experimental Precautions (l) Statistical Methods IV RESULTS A. Effect of Temperature on Germination of Conidia of T. fructigena. 7 14 17 17 17 17 17 18 19 20 20 22 22 23 23 23 24 25 26 30 30 University of Ghana http://ugspace.ug.edu.gh - 5. - Further experiments on the effect of Temperature on the Germination of Conidia of T. fructigena. ... 35 Latent Period of Germination of the Conidia at 20° and 25°C. ... ... ••• 40 Effect of Moderately High Temperature on the Germination and Viability of Conidia of To_ f ructigena. ... ... ... 44 Survival of Conidia of T. fructigena. ... 47 Longevity of Conidia of T._fructi gena stored at 1OOfi> R.H, in Light and Dark, ... ... 72 Longevity of Conidia of T« fructigena stored in water. ... ... ... 76 Effect of loss of water, by Plasmolysis, on Germination Capacity of Conidia of T. fructigena. 79 Further Experiments 011 the Effect of Loss of Water by Plasmolysis on Germination Capacity of Conidia of T. fructigena. ... ... 84 Longevity of Conidia of T. fructigena stored in Sucrose solutions. ... ... 87 Possible Effects of Storage in water on Metabolism T. fruct-igena Conidia. ... ... 92 University of Ghana http://ugspace.ug.edu.gh Page L. Germination of Condia of T, fructigena in aqueous extract of native soil. Mo Cause of Exogenous Dormancy in Conidia of To fructigena in Soil extracte ... 103 No Examination of the Presence of Fungistasis in native soilo . • • • • • • • • 104 0o Possible Volatile Nature of the Fungistatic Principle of native soil. ••• ••• 108 P. Survival of Conidia of Ta fructigena in Extracts of native soil. ••• ••• ••• 1 1 2 Q. Lysis of Conidia of T« fructi^ena in native soil. 116 R. Saprophytic Survival of T. f'ructi^ ena in soil. ... 1 23 So Further Experiments on the Saprophytic colonization of cocoa pod husk buried in soil by T. fructigena. 1 3 0 T. Colonization of cocoa pod husk of increased nutrient content in soil by T« fructi^ena. <#( 1 3 4 Vo GENERAL DISCUSSION ... ... ... 1 4 7 VI. SUMMARY ... ... ... -169 VII. ACKNOWLEDGEMENT # ^ VIII. LITERATURE CITED ... ... ### 1 8 1 IX. A1T0L;"ilaOUS PUBLICATIONS (in chronological order) ... -]89 University of Ghana http://ugspace.ug.edu.gh I. INTRODUCTION Cacao (Theobroaa cacao L.) is attacked by various fungi (Dade, 1932; Urouhart, 196l). The Phycomycetes, Ph.ytophthora palmivqra (Butl.) Butl. and Trachysphaera fructigena Tabor and Bunting, both belonging to the Order Peronosporales, are readily separated from the rest as fruit (pod) parasites. The two fungi have, however, not received equal attention because of three main reasons. First, P. palroTvora causes a far greater loss, about 10 percent annually, (Gregory, 1969), than T. fructigena which destroys only one percent of the annual crop. Dade (1927) placed losses due to T. fructigena at as low as 0.1 percent. Secondly, while T. fructigena, as present knowledge indicates, is restricted to the cacao pod, P. palmivora affects every organ of the plant including the roots and thirdly, T. fructigena is restricted only to West Africa in contrast to the extensive occurrence of P. palmivora in all cacao growing countries (CMI 1952. Map 249; Gregory, 1969). The scant studies which have been made on T. fructigena do not, however, seem reasonable, jince it is generally known that T. fructigena attacks other valuable crops; and control measures would have been more rewarding if they were based on adequate knowledge of the biology of the fungus. T. fructigena is a parasite of banana (Musa spp.) (Brun, 1954; Brun and Merny, 1947; Meredith, 1960; Thorold, 1956), coffee (Coffea spp.) (Bunting, 1923; Morstatt, 1936; Resplandy et al., 1954; Roger et_ al,, 1937; Tabor and Bunting, 1923) - 7. - University of Ghana http://ugspace.ug.edu.gh and Avocado pear (Persea gratissima Gaertn.) (Bunting, 1924). Indeed, the disease on coffee was considered serious enough to be controlled by quarantine regulations. The 1935 Regulation Ordinance applicable to the then Colony and Protectorate of Nigeria, including the Cameroons under British Mandate and the Importation of Plants Regulation Ordinance (1936) for the then Gold Coast prohibited the importation of coffee cherries unless certified to be free from T. fructigena. T. fructigena causes a soft rot of banana fruit and a second fruit disease referred to as Cigar-end, sometimes called Finger-tip, In the Cigar-end disease, infection commences from the perianth. Germ tubes of conidia of T. fructigena germinating on the stigma grow down through the tissues of the style into the blossom-end of the fruit (Leach, 1953, 1954). Infection then slowly spreads along the finger to about 2 cm from the tip, causing the infected portion to change to dark-brown and finally to black, The pulp tissue seems to undergo what may be described as a dry rot and the affected region is sharply delimited from the uninvaded healthy tissue. The disease, in the field, makes no further progress. The rotted end closely simulating a burnt cigar end (Bouriquet, 1959; Ann. Report of the Cameroons Dev. Corp., 1956, 1957, 1958; Brun, 1955). The disease is very prevalent under conditions of high relative humidity (Brun and Champion, 1955). University of Ghana http://ugspace.ug.edu.gh The other disease, fruit rot, is mostly a storage malady that results in a general wet rot of the fruit (Brun, 1955; Meredith, 1960). The damage caused by T. fructigena on bananas in West Africa may be extensive. In the Cameroons (Ann, Report - Dev. Corp., 1958) where banana is a staple food, cigar-end disease of banana caused in 1958 the loss of entire bunches of 10,485 plants. In the preceding three years, 1 9 5 5 - "1 9 5 7, cigar-end disease caused to­ gether by T. fructigena and the fungus, Stachylidium (Verticillium) theobromae, affeoted the entire crop of 65,000, 9 ,000 and 9j000 plants, respectively. In an Annual Report of the Cameroon Development Corporation (1957)? it was stated that control of the cigar-end disease in banana should be sustained for about 9 months of the year. T. fructigena attacks coffee cherries causing blackening and mummification (Resplandy et al., 1954). The disease is referred to as mealypod and was first reported on Liberian coffee (Coffea liberica), in Ghana by Tabor and Bunting (1923). On attack the berries, especially the young ones, turn dark purplish-brown and eventually shrivel and harden. The rotted fruit finally becomes covered with a white or pinkish—brown mealy incrustation of T. fructigena conidia. Entire crop had often been destroyed by the fungus under wet conditions (Mallamaire, 1934). " 9 * - University of Ghana http://ugspace.ug.edu.gh The disease is not confined to the fruits of coffee, for, in 1923 Bunting (1923) found T. fructigena on young shoots of Coffea liberica in Ghana. T. fructigena was found as a wound parasite on Avocado pear in Ghana by Bunting (1923). The extent of damage to this crop has never been estimated. Infected cacao pods become brown in colour, quite similar to early symptoms of P. palmivora infection. T. fructigena attack is soon distinguishable thereafter by the heavy sporulation of the fungus forming a thick pinkish encrusted mealy conidial mass over the surface of the pod. The disease is, therefore, termed mealypod. (See Plates 1, and 2), Infection is commonly through wounds, but the Report on Agricultural Development of the Government of the then Gold Coast for the year 1921, indicated that the organism was sometimes capable of infecting unwounded pods. T. fructigena is undoubtedly economically important. Its significance and importance in West Africa becomes more profound with the usual agricultural practice of mixed farming that naturally encourages spread of the fungus among the various host species. - 10. - University of Ghana http://ugspace.ug.edu.gh T. fructigena is largely a fruit parasite and, therefore, faces the problem of survival between successive fruit-bearing seasons. It is not unlikely that this facultative parasite could exist saprophytically in the soil in the absence of the fruits of the host plants. The conidia produced in large quantities would be especially important as the major agents of dispersal and infection. The literature, however, contains no information both on the survival of the mycelium and on the longevity of the conidia which are likely to be fragile, in common with the asexual propagules of other members of the Peronosporales. In the investigation described below saprophytic survival of T. fructigena in soil and the factors affecting the survival of the conidia were extensively studied to provide valuable and indispensable information for the design of successful control measures. - 11. - University of Ghana http://ugspace.ug.edu.gh - 12. - Plate 1. Photograph showing early stages of development of mealypod on ripened and unripened cacao pods; wound-inoculated with conidial suspension of T. fructigena and incubated at 25°C in humid atmos­ phere for 3 days. (X University of Ghana http://ugspace.ug.edu.gh - 13- - Plate 2. Photograph showing advanced stage of development of mealypod on ripened and unripened cacao pods; wound-inoculated with conidial suspension of T. fructigena and incubated at 25°C in humid atmosphere for k days. Note the encrusted mass of conidia and absence of external mycelium.(X V 3 ) University of Ghana http://ugspace.ug.edu.gh II. LITERATURE REVIEW There are very few references to the biology of T. fructigena in the literature and the little interest so far shown in it might be due to its very limited geographical distribution. The physiology of the fungus can only be inferred from the results of the few inoculation experiments that have been carried out. T. fructigena penetrates better non-fortified host tissues. Tabor and Bunting (1923) observed that mealypod was more serious among young cacao pods because the protective sclerotic tissues had not yet been formed. When Meredith (i960) placed conidia of T. f r u c t i g e n a in wounds made in the skin at the blossom-end and at the mid-region of the fruits of Jamaican bananas, only three of 25 fruits inoculated at the blossom-end were infected while 15 of the 25 fruits inoculated at the mid-region were rotted. In this instance resistance to infection might be due to another cause, since skin tissues at the blossom-end and mid-region of the fruit were apparently of the same thickness and contained identical cell types. Temperature of 15°C was inferior to 21° - 27°C for growth of T. fructigena. Rate of rotting of infected banana fruits was almost doubled at 21° - 27°C (Buxton gt gl., 1962). - 14. - University of Ghana http://ugspace.ug.edu.gh Both conidial formation and sexual reproduction were affected by the growing medium, Tabor and Bunting (1923) detected sexual organs in infected cacao pod husk but could not find any in either the pericarp of diseased coffee or on media containing extract of coffee pericarp. when Meredith (i960) inoculated healthy skin of Jamaican bananas by placing conidial suspension on the intact skin surface, none of the fruits were infected although the conidia germinated very well. The germ tubes were seemingly unable to penetrate the outermost cells of the fruit. His observation, however, contrasted an earlier observation of Brun and Merny (1947), who obtained infection, by similar inoculation procedure, of G-ros Michel banana fruits. Were the conflicting results due to host varietal suscepti­ bility or to varying potency of different isolates of T. fructigena? There is absolutely no information on the survival of the fungus although its seasonal occurrence on banana had been observed in Cameroon. Leach (1953, 1954) reported that at certain times, for example, December, there was abundant evidence of Trachysphaera Cigar-end but little or no Stachylidium Cigar-end; but at other seasons, especially the dry season, Stachylidium was the evident superficial organism. Brun (1955) also observed that the rotting of bananas on certain plantings in Penja and Loum-Chontiers regions - 15. - University of Ghana http://ugspace.ug.edu.gh of Cameroon was very severe between the end of October and December, the disease disappearing almost completely from April to September. The sources of new infections in the wet season in both cases were not investigated. University of Ghana http://ugspace.ug.edu.gh - 17. - III. MJJERL&L AND GENERAL 1JETH0DS (i) Material T. fructigena used was isolated from a naturally infected cacao pod collected from a cacao farm at Aburi. (ii) Genorr.l Methods (a) Spore Germination Tests 1. Slide Method In many of the spore germination tests, the "slide" method was used. Spore suspensions of T. fructigena were prepared by transferring conidia removed from surface of culture on petri plates with a flamed inoculating needle into sterile water in McCartney tubes. The conidia dispersed readily and the suspension needed no shaking. The suspension m s filtered through 4-layers of sterile muslin cloth to remove most of the hyphal fragments. The conidia were then washed free of any nutrients taken up from the petri plates by allowing them to sediment for half an hour in a test tube and discarding the supernatant fluid. The conidia were then re-suspended in sterile distilled water or other germina­ ting medium. On other occasions washed conidia were recovered by centrifuging the suspension at 300 r.p.m. for 5 minutes. University of Ghana http://ugspace.ug.edu.gh The number of spores in suspension for every germination test was strictly standardised to 400 ,000 per millilitre of solution with the aid of a haemacytometer. Sterile petri dishes, each containing a sterile slide (7.5 x 2.5 cm) supported on a glass v-piece, over a small quantity of distilled water had, in the mean time, been equilibrated with the incubation temperature. This technique obviated evaporation of the germination test droplet at the initial stages of incubation. Using an inoculating loop, two individual drops of spore suspension (about 0 . 1 ml in volume) were placed on each thermally equilibrated slide contained in its damp chamber, and the slide immediately returned to the incubation temperature. Each germination count was based on eight drops of suspension (i.e. two separate drops on each of four slides for each treatment). 2. Agar Plate Method Spore germination was also observed on agar media in petri dishes (20 ml of agar medium per dish) in a considerable number of experiments in this investigation. Each agar plate was inoculated with 1 ml of spore suspension divided between 10 evenly spaced areas. The concentration University of Ghana http://ugspace.ug.edu.gh and spatial spread of the spores corresponded closely to what obtained in the "slide" method. (b) Assessment of Conidium Germination At the end of the desired incubation period, the tested conidia were promptly stained with cotton blue (0.1%) in lactophenol. If observations could not be made at once drops of N/ 4 0 formaldehyde were added, in addition to the stain, to make sure that development of the spores was arrested. The Percentage Germination was estimated from a total of not less than 300 conidia from randomly selected fields of the microscope. Any conidium with a discernible germ tube was counted as having germinated. This definition of germination was thus based on morphology. The numbers of conidia examined in each experiment are shown in the tables of data, to show the reliability of the percentage germina­ tion claimed for each treatment. The germ tubes commonly branched so profusely that in most cases it was impossible to make aocurate measurements. Plate 3 demonstrates extensive germ tubes produced by conidia of T. fructigena after only 3 hours incubation on PDA.. The results, therefore, contain no quantitative information University of Ghana http://ugspace.ug.edu.gh on amount of growth of germ tubes obtained and the effect of any treatment was assessed by percentage germination only. (c) Humidity Chambers Transparent plastic boxes, 21.5 cm l°nS> 10.5 cm wide, and 7 . 5 cm deep with tightly fitting lids, served as Relative Humidity Chambers. The lids were usually held firmly in position with cellotape. These boxes proved to be very convenient in that very little condensation occurred at 100fo relative humidity. The humidity chambers were used in experiments on conidial longevity. Microscope slides bearing spore prints of conidia were placed on solid watch glasses standing in the humidity chambers, raising them well above the humidity-controlling solution at the bottom of the plastic chamber. Each humidity chamber held two microscope slides. Water and sulphuric acid solutions used for maintaining constant relative humidities within the chamber were 5 mm deep. (d) Maintenance of Constant Relative Humidity Atmospheres of different relative humidities were obtained and maintained with aqueous sulphurio acid solutions (Solomon, 1952) (see Table 1 ). Nominal zero percent relative University of Ghana http://ugspace.ug.edu.gh - 21. - TABLE 1 Aqueous sulphuric acid solutions for maintaining constant relative humidities at 25°C (Extracted from data of Solomon, M.E., 1952). fo Relative Humidity at 25°C Weight in g of sulphuric acid per 100 g of solution Density at 15°C 20°G 1 00 0 .0 0.999 0.998 95 1 1 . 0 2 1.075 1.073 90 17.91 1 . 1 2 6 1.123 85 2 2 .88 1 . 1 6 5 1 . 1 6 2 80 26-79 1.196 1.193 75 30.14 1.223 1.219 50 43-10 1.334 1.330 25 55.01 1.499 1.445 University of Ghana http://ugspace.ug.edu.gh humidity was maintained with anhydrous calcium chloride, while water provided 100 per cent relative humidity. (e) Spore Prints for longevity tests Preliminary studies have shown that the conidia are extremely sensitive to humidities lower than 100% R.H. Pertinent information has been presented at appropriate places in the text. The following method was therefore devised to obtain spores for longevity tests. A rectangular piece of the culture, about 2 cm x 3 cm, was cut out of the petri plate from a 5 day-old pure culture with a flamed inoculating needle and lifted off the culture with the needle. The culture on the agar block was gently adpressed onto the slide to obtain a spore print. The slide was immediately trans­ ferred into the humidity chamber. Such spore prints were also used in "buried slide" experiments investigating the fate of conidia in the soil. Further details of the "buried slide method" are presented in Chapter Q. (f) jJaintonance of Stnck Culture L stock culture was maintained on slopes of Potato Dextrose jigar Medium (200g. potato; 20g. dextrose; 20g. agar; 1000 ml do-ionised water) in McCartnoy Tubes at 25°C and subculturod fortnightly. University of Ghana http://ugspace.ug.edu.gh (g) Production of Conidia Potato Dextrose Agar supported sufficiently heavy sporulation and was used for that purpose in this work. Spores for use in experiments were consistently obtained from 6 day-old cultures raised in petri plates. (h) Incubation Incubation conditions varied with the experiment and are described at the appropriate places in the text. (i) Aqueous Extract of Soil Extract of soil needed during this investigation was made from soil collected from cocoa farms at Aburi where the disease is prevalent. The profile of the soil showed two distinct layers. There was an upper carpet of partially decomposed leaves, inches thick, overlying a layer of black loam soil. The soil extract was prepared as follows: the soils were moistened for two days (samples from the 2 zones were kept apcrt nnd usod separatoly), and aliquots were \acked into glass tubings, 300 mm long and 20 mm in diameter, with both ends open, though one was plugged with glass wool, before introducing the soil. A tiny glass tubing, University of Ghana http://ugspace.ug.edu.gh 2 mm in diameter, was attached to the bottom of the soil tube (end plugged with glass wool) by a rubber bung, which led the draining fluid into a clean collecting flask. The soil in the glass tube formed a column of 200 mm. Sterile distilled water was then gradually poured by a pipette into the column of soil until it started to drain from the exit tube, and then an equal volume of the water so far introduced was added to the column. The extraot collected at intervals was poured onto the soil column to drain through again. The process was continued for 3 hours. All extracts were used immediately after preparation. (j) Methods of Sterilization A H glass-ware and plastic containers were soaked in solutions of teepol, thoroughly washed, rinsed.under running tap water and thoroughly rinsed in distilled water. A H media, medicinal flats, McCartney tubes, conical flasks, pint milk bottles, capped test-tubes and muslin filters, were sterilized by autoclaving for 15 minutes at 15 lbs per square inoh steam pressure. Non-absorbent cotton wool plugs of culture vessels were temporarily K'caf't covered with #roaac^ paper to prevent the penetration of any condensed water during autoclaving. University of Ghana http://ugspace.ug.edu.gh - 25. - Petri-dishes and pipettes were sterilized by- heating at 160°C for 6 hours in an electrically heated oven. Inoculation needles, spatulae and microscope slides were flame sterilized. The microscope slides were stored in 9C0S ethyl alcohol, and flame sterilized just before use. Surface of cacao pods were sterilized with 95% alcohol. The inoculation room was sterilized by spraying with 3$ dettol solution and kept closed for 1 0 minutes just before use. (k) Experimental Precautions 1. Sulphuric acid solutions were prepared with caution. Care was taken to avoid generation of heat too quickly. 2. Soil for the preparation of extraots was not allowed to dry after collection from Aburi. They were University of Ghana http://ugspace.ug.edu.gh - 26. - kept in plastic bags and regularly watered. 3. Spore print preparation was carried out with petri plates and humidity chambers under a hood of plastic sheet to prevent exposure of the conidia to dry air. 4. Spores of the same age were used in all experi­ ments to prevent erratic results. (l) Statistical Methods Correlation and Regression 1, In the assessment of correlation between percentage of collapse and percentage of germina­ tion of conidia, the basic observations were n pairs of associated observations represented by (x, y) for % collapse and % germination, respectively. The assumption was that x and y followed, at _ . n ortnai least approximately, a bivariate^distribution. The University of Ghana http://ugspace.ug.edu.gh correlation coefficient r was given by the equation: ^ _ Covariance x y _ \ Variance x. Variance y which is the same as r = ^ X ~ ^ ^ = M = - x)2. £ (y - y)2 To facilitate the use of calculating machines, the equivalent equation shown below was used: ix . £ y ~ n ____________ ^ ( ^ x 2 - £ 2 x /n ) . ( £ y 2 - S.2 y /n) When it was found necessary, the r values were converted to the student's t-test values by the following equation: r ^ n - " 2 M 1 - r 2 2. For the Regression, the true regression line for the regression of y on x is known to be: y = a + |&x. The true regression coefficient j2> was first estimated by the equation: = K * - * H y - y ) £ ( x - x ) 2 which is the same as its equivalent and convenient equation below: £ x ■ gy b 2 *y - n £x2 - I 2 X/n - 27. - University of Ghana http://ugspace.ug.edu.gh - 28. - The constant a was calculated by the equation: a = y - b x. The fitted regression line was then as follows: y = a + bx. Coefficient b was only an estimate of the true regression coefficient and so, it was necessary to prove that it was a good estimate of ^ . Since n was ^30, the normal test (where if b is more than 1.96 standard errors away from zero, then it is significant at the 5% level) was used. The standard error (s.e.) of b was calculated from the equation: Using the calculated constant a and regression coefficient b two values of x were calculated, one from a given maximum value of y and another from a given minimum value of y. The two points were connected with a straight line to form a regression line on a graph, where the x axis shows percentage collapse and the y axis shows percentage germination. The information was obtained from Bailey's Statistical Methods in Biology (Bailey, 1959). b + s.e where variance S',2 [ * ( x - x ) ( y - yj £.( x - x ) 2 University of Ghana http://ugspace.ug.edu.gh - 29 - - Plate 3* Photomicrograph of germinating conidia of T . fructigena incubated on Potato Dextrose .Agar at 25°C for 3 hours. (X 120) University of Ghana http://ugspace.ug.edu.gh IV. RESULTS A. Effect of Temperature on Germination of Conidia of T. fructigena The major portion of this investigation examined the longevity of the conidia. It was considered important to determine the germination capacity of the conidia after various storage treatments at the optimum temperature, where germination activity would be maximum. The germination of conidiosporangia of several members of the Peronosporales has been studied. In most of these species the phenomenon of germination differs from that of T. fructigena. The conidiosporangia germinate either directly by producing germ tubes or indirectly by means of zoospores. Germination of T. fructigena conidia is comparable with direct germination of these species. The conidiosporangia, in most instances, produce zoospores at considerably low temperatures with an optimum temperature below average tropical atmospheric temperatures. Germ tubes, on the other hand, are formed at higher temperatures. To illustrate^ Clerk (1972) reported that indirect germination of sporangia of Phytophthora palmivora in distilled water was optimal at 22°C over the range 10 to 34 C while direct germination occurred only at 30° and 34°C. Wolf and Wolf (1947) reported an optimum temperature of 12 - 13 C for indirect germination of sporangia of University of Ghana http://ugspace.ug.edu.gh - 31 - Phytophthora infestans, and best germination, by means of germ tubes, at 24°C. Aragaki et al. (1967), who did not obtain any direct germination in water in Phytophthora parasitica sporangia, found a comparatively higher optimum temperature of 24 - 28 C for indirect germination. The behaviour of the conidiosporangia could, however, be influenced by other factors, particularly nutrients. Thus Clerk (1972) found that sporangia of P. palmivora germinated well by means of germ tubes at 22°C in peptone and yeast extract solutions and in the extract of cocoa pod husk. Similarly, Aragaki et al. (1 9 6 7) reported that papaya extract supported the best direct germination of 51 percent at 28°C. In finding the optimum temperature for germination of the V conidia of T. fructigena to be used in subsequent experiments, un ifo rm ity i* not e-xpee-ted, the results would also indicate, although^^ !^— whether its optimum temperature is close to that for direot germination of other species of the Peronosporales. The slide method was used. Aqueous conidial suspension drops were Incubated at 15°, 20°, 25°, 30°, J5° and 40°C. There were four slides at each temperature and observations were made on the same suspension drops over the entire period. The slides were withdrawn at 6, 12, 2 4, 36 and 48 hours; for counts of University of Ghana http://ugspace.ug.edu.gh - 32. - germinated conidia to be quickly made and returned to the incubators. The results are presented in Table 2. The results show that germination was poor in distilled water. There was no germination at ifO°C, even after 48 hours of incubation. Germination was clearly very low at 35°C. There was only 0.5 percent germination at 35°C, compared to 1.4, 1.4, 1.2 and 1.1 percent respectively, at 15°, 20°, 23° and 30°C after 48 hours. Although percentage germination was practically the same at 15° - 30°C, the germ tubes were far shorter at 30°C than at 15° - 25°C. University of Ghana http://ugspace.ug.edu.gh - 33 . - TABLE 2 Germination of conidia of T. fructigena incubated in distilled water at different temperatures for 48 hours. Temperature of incubation °c Period of incubation in Hours Total number of conidia observed Percentage Germination 6 393 0.3 12 410 1.7 15 24 413 1 . 2 36 406 1 . 2 48 414 1.4 6 410 0.5 1 2 445 0.5 20 24 452 1 . 1 36 468 1.3 48 442 1.4 6 385 0.8 1 2 406 1 . 2 25 24 423 1 . 2 36 434 1 . 2 48 416 1.2 University of Ghana http://ugspace.ug.edu.gh - 34 . - TABLE 2 (cont'd.) Germination of conidia of T. fructigena incubated in distilled rnter at different temperatures for 48 hours. Temperature Period of Total of ' incubation number Percentage incubation in of conidia Germination o^ Hours observed 6 385 1.3 1 2 392 1.3 30 24 504 1 . 0 36 453 1.1 48 457 1.1 6 459 0 .2 1 2 423 0 .2 35 24 452 0.4 36 392 0.5 4B 412 0.5 6 354 0.0 12 398 0.0 40 24 405 0 .0 36 409 0 .0 48 3 7 1 0.0 University of Ghana http://ugspace.ug.edu.gh - 35. - B. Further experiments on the effect of Temperature on the Germination of Conidia of T. fructigena It was observed in the previous experiment that germination of the conidia of T. fructigena was best favoured at 15°, 20° and 25°C. The amount of germination in water was, however, so low that it was not possible to make any reliable comparison. The conidia were, therefore, germinated again, using in this instance PDA medium. The same range of temperature of 15° to 40°C, was again used in this experiment. The temperature levels of 35° and 40°C which were clearly unsuitable in the distilled water experiment were included to find out whether germination would be improved in the presence of extraneous nutrients. The 'Agar Plate Method' was used and for each temperature, assessment of percentage germination was again made after 6, 12, 24, 36 and 48 hours. There were four petri plates for each temperature. The results are presented in Table 3* There was again no germination at 40°C. Germination at some of the temperature levels was, however, highly stimulated. At 12 hours, the last recording time for 20° and 25°C, percentage germination was 94.0 and 96 .5 percent at 20° and 25°C, respectively and 33.6 and 30.2 percent at 15° and 30°C, respectively, for the same period, iilthough almost the same amount of germination was University of Ghana http://ugspace.ug.edu.gh - 36. - TABLE 3 Germination of oonidia of T. fructigena on Potato Dextrose Agar at different temperatures for 48 hours. Temperature of incubation °c Period of incubation in Hours Total number of conidia observed Percentage Germination 6 421 13-4 12 440 33.6 1 5 24 404 35.1 36 417 45.8 48 408 52.7 6 359 87.2 12 400 94.0 20 24 * --- 36 ---- --- 48 ---- --- 6 373 96.8 12 428 96.5 25 24 _ — --- 36 — — ---- 48 ---- ---- ^Observations were discontinued because the germ tubes developed so profusely that their origin could not be traced while some hyphae began to produce conidia after 36 hours. University of Ghana http://ugspace.ug.edu.gh TABLE 3 (cont'd.) - 37 . Germination of conidia of T. fructigena on Potato Dextrose Agar at different temperatures for 4-8 hours. Temperature of incubation °G Period of incubation in Hours Total number of conidia observed Percentage Germination 6 305 34.4 1 2 334 30.2 30 24 429 2 6 . 1 36 -- + ---- 48 --- ---- 6 429 0 .0 12 418 0 .0 35 24 418 0 .2 36 397 0.3 48 — - --- 6 382 0 .0 12 441 0 ,0 40 24 390 0 .0 36 395 0 .0 48 420 0 .0 Although germ tubes were discernible, new conidia were produced and so observations were discontinued. University of Ghana http://ugspace.ug.edu.gh eventually attained at 12 hours, at 20° and 25°C, rate of germination was slightly faster at 25°C. At 6 hours, there was 96.8 percent germination at 25° C and 87.2 percent at 20°C. The optimum temperature was apparently 25°C and was adopted for subsequent experiments. The poor germination at 35°C, 0.3 percent germination at 36 hours, indicated that the conidia might be rather sensitive to higher temperatures. It was not possible to extend reading beyond this period for further observation of the conidia because of the production of conidia by the germ tubes (Fig. 1 ). University of Ghana http://ugspace.ug.edu.gh - 39. F I G . ! D E V E L O P M E N T OF C O N I D I A BY G E R M I N A T I N G T. F R U C T I G E N A C O N I D I A I N C U B A T E D ON P D A A T 2 5 ° C A N D 3 0 ° C F O R 3 6 H O U R S . A , P R I M O D I A L CON I D I A B, N EW D E V E L O P E D CON I D I A C, O L D C O N I D I A University of Ghana http://ugspace.ug.edu.gh C. Latent Period of Germination of the Conidia at 20° and 25°C Many criteria are used in assessing the effect of environ­ mental factors on spore germination. These mainly are, estimating percentage germination after a specific time lapse; noting time for the first spore to produce a germ tube and, measurement of the time taken by 50 percent of the germinable spores to produce germ tubes. This last criterion, referred to as latent period of germination, as well as the second method, enables us to differen­ tiate clearly between two treatments with closely similar effects, especially when percent germination are almost identical at the end of the incubation period. The latent period of germination also provides information on the rate at which the final percentage germination was achieved. It was found in the previous experiment (see Chapter B) that germination was very high after 12 hours at both 20° and 25°C, although 25°C was slightly superior. An experiment was carried out to estimate percentage germination at hourly intervals and thus the rate of germination at the two temperatures, to bring out more clearly the effects of these two temperatures on the germination of the conidia. University of Ghana http://ugspace.ug.edu.gh The conidia were germinated on PDA, using the ’Agar Plate Method1 over 8 hours. There were four petri plates at each temperature and hourly observations were made on the same plates throughout the experiment. The results, percentage germination for each hour, are recorded in Table 4 and graphically represented in Fig.2. The results (Table 4) show that the rate of germination of conidia of T. fructigena was faster at 2p°C. The latent period of germination at 25°C was, as calculated from the graph, 96 minutes while that at 20°C was 150 minutes. The earliest germina­ ting spores at 20° and 25°C were observed after 56 and 32 minutes, respectively. Germination was almost completed after 3 hours at 25 C and after 4 hours at 20°C. Again, the final percentage germination was slightly higher at 25°C. University of Ghana http://ugspace.ug.edu.gh - 42 . - TABLE 4 Germination of conidia of T, fructigena incubated on PDA at 20° and 25°C for 8 hours. Temperature of incubation •c Period of incubation in Hours Total number of conidia observed Percentage Germination 1 352 1.4 2 375 22.7 3 380 66 . 1 20 4 386 80.6 5 402 83.4 6 369 86 .4 7 359 83.0 8 360 84.7 1 363 1 6 . 8 2 369 65.O 3 374 86 .7 25 4 415 90.2 5 337 87.7 6 385 90.8 7 379 91.0 8 338 88 .4 University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E OF G ER M IN A T IO N - 43. - FIG. 2 GERM INAT ION OF CON ID IA OF T. F R U C T IG EN A I N C U B A T E D AT 2 0 ° AND 2 5 ° C . 2 0 0 C o----------- o 2 5 ° C University of Ghana http://ugspace.ug.edu.gh D. Effect of Moderately High Temperatures on the Germination and Viability of Conidia of T. fructigena No germination occurred at 40°C in both water (see Table 2) and on PDA (see Table 3)« T. fructigena clearly appears to be non-thermophilic. It is necessary to find out whether the conidia were killed by the relatively moderate temperature of 40°C within 12 hours or merely prevented from germination. There are instances where spores of other fungal species that have not germinated at such temperature have done so on transfer to lower temperatures. For example, Frossard (1962) observed that, ascospores of Mycosphaerella musicola, the causal agent of Sigatoka disease of banana, which do not germinate beyond 38° - 4-0°C, were capable of germination at the optimum temperature for germination after they had been placed in a saturated atmosphere at 40° and 42°C for 21+ hours. The spores we re, however, killed by 6 hours exposure to 44°C and after storage at 50°C for only one hour. This experiment was carried out to find out the extent to which T. fructigena conidia could withstand slightly more than normal tropical temperatures, which are not unlikely to occur in a vigorously respiring cocoa pod or in decomposing leaf litter on the ground. University of Ghana http://ugspace.ug.edu.gh Sixteen petri plates of PDA with spore suspension drops were incubated at 4.0° and another similar set at 45°C. Four plates were withdrawn from each temperature at 3, 6, 12 and 1 6 hours and placed at 25°C for 12 hours. The percentage germination of the conidia was then assessed at the end of this period. The results are presented in Table 5* The results show that there was no germination after incubation at 1+5°0, even for as short a period as 3 hours. Some germination was obtained in conidia kept for 12 hours at 4-0°C. Survival was, however, very low, 0.5 percent, and most of the conidia would be killed by this temperature in less than 12 hours. Only 28.5 percent of the conidia were viable after 6 hours1 exposure to 4-0°C. University of Ghana http://ugspace.ug.edu.gh - 4 6 . - TABLE 5 Germination of conidia of T. fructigena stored on PDA at 40° and 45°C for varying periods (HOURS) and then transferred to the optimum temperature (25°C) for 12 hours. Temperature of storage °c Period of storage in Hours Total number of conidia observed Percentage Germination 3 401 52.1 6 432 28.5 40 12 397 0.5 18 399 0.0 3 348 0.0 6 334 0.0 45 12 342 0.0 18 336 0.0 University of Ghana http://ugspace.ug.edu.gh - 47. - E. Survival of Conidia of T. fructigena The length of life of fungal spores, as measured by their ability to germinate after various periods, is affected, in the main, by environmental conditions of humidity, temperature and light. At any given relative humidity, increasing the temperature generally decreased the viability of fungal spores, and lower temp­ eratures, above freezing, favour longevity (Cochrane, 1958; Ainsworth and Sussman, 1968). Fergus and Schein (i960) found that light had a deleterious effect on viability of stored ascospores of Vinula craterium, and spores exposed to light died quicker than those kept in darkness. Clerk and Madelin (19 6 5) also observed that Mptarrhizium anisopliae conidia lived longer in darkness particularly when the spores were stored at 8°C. The relationship between relative humidity and viability is not simple, and four main sorts of relationship have been reported, and have been recently fully reviewed by Ainsworth and Sussman (1968), (1) Several investigators have found that lower relative humidities favour retention of viability of specific fungus spores, (deBruyn, 1926; Harrison, 1942; Maneval, 1924; MoCrea, 1923; McKay, 1935). University of Ghana http://ugspace.ug.edu.gh - 48. - (2) Hart (1926) reported that uredospores of Melampgora lini retained their viability for a longer time at mid-humidities (40% and 6CP/o R.H.) than at higher and lower humidities, while Rosen and vfeetman (1940), and Naqvi and Good (1957), respectively, found similar behaviour in uredospores of crown rust of oats and conidia of Monilinia fructicola. (3) Merek and Fergus (1954) found that at 12° - 24°C, the endoconidia of Endoconidiophora fagacearum remained viable longer at 95% than at 75%> R.H., and Goos and Tschirsch (1962) reported that spores of Gloeosporium musarum survived longest at higher humidities (6C% - 8C% R.H.) than at lower humidities (C% - 20% R.H.). Hyre and Cox (1953) found that at 20° and 30°C sporangia of Phytophthora phaseoli did not germinate in any saturated atmospheres, unless the sporangia were in free water. At 20°C, nearly 40 percent sporangia were viable after 27 hours exposure to 93% or 100% R*H. and 25 percent were viable after 50 hours. They found that longe­ vity decreased rapidly at lower humidities - less than 10 percent was viable after 4 hours, 1 hour and 15 minuties at 9C%>, 86% and 81% R.H., respectively. Fewer sporangia were viable after exposure at corresponding humidities and periods of time during storage at 30°C. Less than 10 percent were viable at 93%, 97% and 10C% R.H. after 2? 10 and 27 hours, respectively. University of Ghana http://ugspace.ug.edu.gh (4) Teitell (1958) reported that viability of conidia of Aspergillus flavus was preserved longest at Ofo and Q /^o R.H., and lost quickest at 73f° R.H. The only other observation of this response was that of Clerk and Madelin (19 6 5) w^° that conidia of Metarrhizium anisopliae survived longest at the extreme relative humidities, ^ - 3 R.H. and 8^0 - 99?° R.H. and lived shortest at the median humidities, 55$ ~ R.H. Teyegaga and Clerk (1972) found that the response of conidia of Cercospora canescence to storage humidity was different in light and in dark. In the dark, the conidia lived longest at the lower humidities, Ofo - 2Qf/o R.H. and longevity shortened with increasing relative humidity. In light, however, humidities of 20% to !+Oft> R.H. were the most favourable for survival, with the conidia dying quickest at Cfi R.H. and 60% - QCF/o R.H. An experiment was carried out to find the effect of relative humidity - which would be the most important climatic factor on cacao farms - on the survival of T. fructigena conidia. Atmospheric temperatures vary very little (Boateng, 1960), the range being on the average, 26.1° to 28.9°C annually for Ghana. Information from three experiments - the results of whioh have not been quoted in full since no germination occurred in any - preceded this major survival experiment. The experiments were briefly as follows:- - 49. - University of Ghana http://ugspace.ug.edu.gh 1. The conidia did not germinate at 8C^ > - 100$> R.H. 2. The conidia did not survive 24 hours storage at O/o - 8Cfo R.H. 3. In a second experiment the conidia did not survive 6 hours storage at Cfo - 80% R.H. 4. In a third experiment no conidia survived one hour storage at Cfo - 8($> R.H. Every experiment was repeated three times. T. fructigena conidia were apparently very sensitive to drying and a major experiment was set up using 75ft> - 10C% R.H. as the storage humidity and observations of their viability commencing after only 20 minutes of storage. Spore prints for longevity tests were prepared and immediately transferred into the humidity chambers, as outlined in sections (c), (d) and (e) of General Methods. The spores were placed in plastic chambers with constant internal humidities of QCffo, 89%, and \OCf/0 R.H. and incubated at 25°C in incubators with no internal lighting. Samples from each humidity level were withdrawn at 20 minutes intervals for the first two hours of incubation and then, at hourly intervals until the 6th hour of incubation, followed by germination - 50. - University of Ghana http://ugspace.ug.edu.gh tests at 9, 12, 21+ hours and, thereafter, after the number of days of storage indicated in Tables 10 and 11, which concerned those stored at and lOOfc R.H- only. On withdrawal after storage, the spores on the slides were washed off with sterile distilled water to make a spore suspension which was used in seeding petri plates of PDA. The inoculated petri plates were then incubated at 25°C for 12 hours and the percentage germination, and hence percentage viability, assessed. VThenever zero percent germination was recorded, germination tests were made on the two succeeding sampling times to confirm total death earlier recorded. In the earlier experiments outlined above, the conidia were found to collapse or shrink at all the humidities between Ofo and 80^ R.H. Normal conidia and shrunken conidia are shown in Plates 4 and 5, respectively. Perhaps shrinkage was related to death of the spore and in all the present tests, slides selected at random from each humidity level were examined under the microscope and the number of spores which had collapsed was quickly estimated. These data are included in the results presented in Tables 6 to 11 and graphically represented in Figs. 3, 4 and 5. The results in Tables 6 and 7 show that the conidia died very quickly at 7 and at 80^ R.H., and the conidia survived for University of Ghana http://ugspace.ug.edu.gh - 52. - TABLE 6 Survival of Conidia of T. fructigena stored at 75% a-t 25 C Period of' storage in hours Estimation of Percentage Estimation of of conidia that collap­ sed in storage Total number of conidia observed Percentage collapse Percentage germi­ nation of viable conidia on PDA after 12 hours Total number of conidia observed Percent- Germi­ nation 3 296 97.6 382 0.5 2 3 309 99.0 422 0.0 1 298 99.3 419 0.0 284 99.3 381 0.0 University of Ghana http://ugspace.ug.edu.gh - 53 TABLE 7 Survival of Conidia of T. fruotigena stored at 8C$ R.H. at 25°C Period of storage in hours Estimation of Percentage of conidia that collapsed in storage Total number of conidia observed Percentage collapse Estimation of Percentage germi­ nation of viable conidia on P M after 12 hours Total number of conidia observed Per cent- Germi­ nation 1 3 285 95-1 385 1.6 2 3 284 95.8 395 0.5 1 289 98.6 386 0.0 286 98.3 365 0 .0 1-1 286 100.0 397 0 .0 University of Ghana http://ugspace.ug.edu.gh - 54. - TABLE 8 Survival of Conidia of T. fructigena stored at 85/o R.H. at 25°C Period of storage Estimation of Percentage of conidia that collapsed in storage Estimation of Percentage germi­ nation of viable conidia on Pm after 12 hours in hours Totalnumber of conidia observed Percentage collapse Total number of conidia observed Percent­ age Germi­ nation 3 296 86.8 397 4.3 2 3 285 94.4 387 0.8 1 278 97.1 392 1.0 1* 284 93.3 365 1.1 if 285 99.3 393 0.5 2 265 99.2 384 0.0 3 269 99.6 381 0.8 4 261 99.2 384 0.8 5 248 96.8 377 0.3 6 279 97.5 366 0.6 9 280 100.0 382 0.3 1 2 271 95.2 383 0.0 24 268 99.3 382 0.0 48 301 100.0 354 0.0 University of Ghana http://ugspace.ug.edu.gh - 55. - TABLE 9 Survival of Conidia of T. fructigena stored at 9C$ R.H. at 25°C Period of storage in hours Estimation of Percentage of conidia that collapsed in storage Total Percentage number collapse of conidia observed Estimation of Percentage germination of viable conidia on PD& after 12 hours Total Percentage number germination of conidia observed 281 2 3 .8 343 43.1 2 3 277 48.0 385 25.5 1 282 81.6 408 6.1 i£ 272 93-4 369 10.3 1* 253 97.3 387 1.6 2 265 97.0 390 2.6 3 275 80.0 434 10.1 4 273 86.1 399 12.8 5 261 82.0 379 9.0 6 261 92.7 369 4.6 9 258 93.0 378 2 .4 12 281 90.7 374 0.3 24 274 98.9 373 0.0 University of Ghana http://ugspace.ug.edu.gh - 56 . - TABX* 9 (cont'a.) Survival of Conidia of T. fructigena stored at 90% R.H. at 25°C Period of storage in Days Estimation of Percentage of conidia that collapsed in storage Total Percentage number collapse of conidia observed Estimation of Percentage Germination of viable conidia on PDA after 12 hours Total Percentage number Germination of conidia observed 2 275 94.2 376 0.3 3 270 98.2 390 0.0 4 250 95.6 391 0.0 5 282 93.3 383 0.0 University of Ghana http://ugspace.ug.edu.gh - 57. - TABLE 10 Survival of Conidia of T. fructigena stored at R.H. at 2 5°G Period of storage in hours Estimation of Percentage of conidia that collapsed in storage Total Percentage number collapse of conidia observed Estimation of Percentage Germination of viable conidia on PDJl after 12 hours Total Percentage number Germination of conidia observed 3 279 3.2 396 92.2 2 3 300 5.0 359 83.0 1 258 22.1 359 75.8 ii 270 19.3 379 81.0 if 264 2 4 .2 381 51.2 2 303 34.3 422 41.7 3 252 4 6 .0 422 71.1 4 274 2 5 .2 378 61.9 5 276 21.7 386 8 1 .6 6 316 26.7 358 71.2 9 294 23.8 337 77.7 12 260 22.3 413 65.4 24 259 49.8 434 33.2 University of Ghana http://ugspace.ug.edu.gh TABLE 10 (cont'd.) - 58. Survival of Conidia of T. fructigena stored at 93$ R.H. at 25 C Estimation of Percentage Estimation of Percentage of conidia that collapsed Germination of viable in storage conidia on PDA after Period of 12 hours storage in Total Percentage Total Percentage Days number collapse number Germination of of conidia conidia observed observed 2 291 68.7 369 10.3 3 291 90.7 379 5.3 4 308 91.1 396 20.7 5 257 88.9 375 11.7 10 272 90.4 365 0.0 15 298 99.3 387 0.0 20 319 100.0 358 0.0 University of Ghana http://ugspace.ug.edu.gh - 59. - TABLE 11 Survival of Conidia of T. fructigena stored at 10($> R.H. at 25°C Period of storage in hours Estimation of Percentage of conidia that collapsed in storage Total Percentage number collapse of conidia observed Estimation of Percentage Germination of viable conidia on FDA after 12 hours Total Percentage number Germination of conidia observed 1 3 245 1 . 6 396 94-7 2 3 304 2.3 382 97.1 1 297 2.7 392 95.9 ii 273 1.8 369 95.9 if 269 4.5 381 93.4 2 282 11.7 395 89.9 3 269 3.7 397 91.9 4 267 2.3 365 87.1 5 270 11.9 373 95-7 » 273 15.4 371 91.9 9 303 5.0 389 97.4 12 266 2.6 409 94-1 24 277 15-5 366 90.2 University of Ghana http://ugspace.ug.edu.gh - 60. - TABLE 11 (cont'd.) Survival of Conidia of T. fructigena stored at 100% R.H. at 25 C Estimation of Percentage Estimation of Percentage of conidia that collapsed Germination of viable in storage conidia on PDA after Period of 12 hours storage in Total Percentage Total Percentage Days number collapse number Germination of of conidia conidia observed observed 2 267 14.6 364 91.5 3 259 2 0 .8 411 82.7 4 297 33-0 424 88.4 5 280 6 .8 403 93-5 10 279 1 1 . 8 389 90 .0 15 26 1 16.5 409 66.7 20 293 49.8 330 29.7 30 283 99.3 336 14.9 40 261 10 0 .0 352 0 .6 50 272 98.2 341 0 .0 60 285 99.3 346 0 .0 70 269 10 0 .0 331 0 .0 University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E C O L L A P S E P E R C E N T A G E GE RM I N A TI 0 N T I M E OF STORAGE IN HOURS FIG. 3 EFFECT OF RELATIVE HUMIDITY ON LONGEVITY OF CONIDIA OF T. FRUCTIGENA STORED IN LIGHT AT 25°C m-------- m 7 5 % R . H . A---------d. 9 0 % R.H. □ a 8 0 % R.H. * ............ 9 5 % R.H. ▲----------A 8 5 % R-H. o---------- o 1 0 0% R.H. University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E C O LL A P S E _ P E R C E N T A G E G E R M IN A T IO N T I M E OF S TORAGE IN HOURS FIG.4 EFFECT OF RELATIVE HUMIDITY ON LONGEVITY OF CONIDIA OF T. FRUCTIGENA STORED IN LIGHT AT 25°C - ■ 7 5 % R.H. - A 9 0 % R.H. - □ 8 0 % R.H. 9 5 % R. H. — A 8 5 % R.H. o 1 0 0% R.H. University of Ghana http://ugspace.ug.edu.gh 100 University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E G E R M I N A T I O N 100 University of Ghana http://ugspace.ug.edu.gh only 20 and 40 minutes, respectively. At 85% R.H. (Table 8), some conidia (Q .% ) were viable after 9 hours but none after 12 hours. This humidity level was unfavourable for conidial survival was below one percent after 80 minutes. The conidia of T. fructigena v/ere clearly extremely sensitive to drying, for at 90% R.H. (Table 9) more than 50 percent of the spores died in 20 minutes and all lost viability between 5 and 10 days at 99$ R.H. (Table 10). They were only well preserved under very high humidity conditions (Table 11). Survival at 100% R.H. was 90.0 percent after 10 days and 29.7 percent after 20 days while a few (0.6%) were still viable after 40 days. There seemed to be a strong relationship between death and shrinkage. This was clearly visible in Tables 10 and 11, where the conidia lived sufficiently long. It was, therefore, decided to test the relationship by means of a correlation coefficient r and to graphically represent the data by means of a regression line. Since one of the most noteworthy assumptions in calculating correlation coefficient is that the distribution of scores must display at least a rough approximation to the normal curve or linear plot, a scatter diagram was plotted on a graph to determine whether there was an apparent degree of association or correlation between conidial collapse and viability (see Pig. 6). Vjhen it was clear that the association was apparent, the correlation coefficient r was calculated according to the statistical procedure outlined in the General Methods, - 64. University of Ghana http://ugspace.ug.edu.gh - 65 . - section (l) 1, using the data of Tables 8 to 11, excluding the figures which had zero percentage germination. Experimental r was = -0.989. The theoretical level of significance when n = 62, was r = + 0.2J0 and at the 0.1% level r was = + 0.408. Since 0.989 ^ >0.408 >0.250, the experimental r was found significant even at the 0.1% level of significance. Probability P. was 0.0001. Converted to the t- test, experimental t was = 51*764, and the level of significance t = 1.95, while 0.1% level t = 3.48. Since 51.764 >3*48 >1.95, then correlation coefficient r was significant, indicating a high degree of negative correlation between collapse and viability. ii regression line was drawn calculated according to the method in section (l) 2, (General Methods). The Standard Error of b when n = 62 was found to be: b + s.e. = -0.9885 + 0.0334, showing b to be 29.6 Standard Errors away from zero. Since 29.6 > 1 .96, then it was significant and so, b was regarded as a good estimate of the true regression coefficient U and was used to calculate and draw the University of Ghana http://ugspace.ug.edu.gh regression line y = 95»71 _ 0 . 9885x which is graphically represented in Fig.6. It is clear from the result in Fig.6 that an increase in percentage collapse was associated with a decrease in percentage viability. A few remarkable features of the shrunken conidium are worthy of note. The conidium collapsed in a characteristic way; one hemisphere of the spore sank into the other hemisphere simulating a deflated ball with one side pushed in (Plate 5)« When a shrunken conidium was immersed in water, the dead conidium assumed again its spherical shape. Such a conidium could, however, be readily distinguished from a living conidium by its dense rounded cytoplasmic mass well withdrawn from the spore wall (Plates 6 and 7)• University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E G E R M IN A T IO N - 67 . - FIG. 6 CORRELAT ION BETWEEN PERCENTAGE COL LAPSE AND PERCENTAGE SURVIVAL OF CON ID IA OF T. F RUCT IG ENA STORED AT VARIOUS RE LAT I VE HUMIDIT IES AT 2 5 ° C ( DATA FROM T A B L E 8 TO II ) • , 8 5 % R . H . a , 9 0 % R. H. A , 9 5 % R . H o , 1 0 0 % R . H . University of Ghana http://ugspace.ug.edu.gh Plate 4. Photomicrograph of unstained conidia of T. fructigena maintained in water. (X 400) University of Ghana http://ugspace.ug.edu.gh - 69. - Plate 5. Photomicrograph of unstained conidia of T. fructigena which have shrunken on exposure to the atmosphere on a dry slide. (X 400) University of Ghana http://ugspace.ug.edu.gh - 70. - w i W € Plate 6. Photomicrograph of unstained dead shrunken conidia of T. fructigena which have rounded up again on immersion in water. Note the central rounded cytoplasmic mass. (X 4-00) University of Ghana http://ugspace.ug.edu.gh - 71. - Plate 7. Photomicrograph of five unstained living conidia of T. fructigena in water and five dead shrunken conidia of T. fructigena which have rounded up again on immersion in water, identified by the central rounded cytoplasmic mass. (X 400) University of Ghana http://ugspace.ug.edu.gh F. Longevity of Conidia of T. fructigena stored at lOCfc R.H. in Light and Dark The experiments on survival had been carried out at a single temperature of 25° C and in incubators which did not have internal lighting* It is reasonable to presume that the spore viability would follow the common trend in relation to temperature. That is, lower temperature would allow a slow rate of metabolism and so prolong the life of the spore, while higher temperatures would shorten it by hastening metabolism, with attendant accumulation within the spore - of deleterious compounds. The important aspect of the temperature relationship, therefore, for each individual species, is the extent of decrease of longevity with temperature rise. The conidia were incubated at 10C% R.H., at 20°C(air-conditioned room) and 28°C (laboratory), where light and dark treatments could be given, over 20 days. Light was provided by combined day-light and fluorescent light to give a light intensity of 2 7 5 .0 and 4^7*5 lux at 20° and 28°C, respectively. Two sets of spore prints were prepared for each temperature, one set to be kept in light and the other in dark. Those for dark treatment were contained in plastic chambers covered with black photographic paper. Samples were withdrawn after 5, 10, 15 and 20 days and their viability assessed by germinating them on PDA for 12 hours. The results are presented in Table 12 and in Fig.7. University of Ghana http://ugspace.ug.edu.gh - 73. - TABLE 12 Longevity of Conidia of T. fructigena stored at 10C% R.H. in light and dark and at different temperatures over 20 days. Temperature (°C) Light Treatment Period of storage in Days Total number of conidia observed Percentage Germination 5 379 79.9 Light 10 360 84.7 (2 7 5 .0 lux) 15 359 39.0 20 355 25.9 20 5 370 74.9 Dark 10 331 81.9 15 385 56.9 20 364 20.6 5 364 71.4 Light 10 329 21.9 (467.5 lux) 15 350 21.4 29 367 17.7 28 : 1 5 358 73.2 Dark 10 328 31.7 15 327 21.0 20 353 15.9 University of Ghana http://ugspace.ug.edu.gh PE RC EN TA G E G ER M IN AT IO N _ PE RC EN TA G E G ER M IN A TI O N OO r 80 5 10 15 20 P E R I O D O F S T O R A G E I N D A Y S FIG. 7 LONGEVITY OF CONIDIA OF T.FRUCTIGENA STORED AT 100% R.H. IN LIGHT AND DARK AND AT 20°C, 28°C o o S T O R E D I N L I G H T • --------------- • S T O R E D I N D A R K University of Ghana http://ugspace.ug.edu.gh - 75. - The results (Table 12) show that some conidia were still viable after 20 days at both temperatures. **1'though conidia in dark and light showed closely similar amounts of viable conidia at the end of the experiment, thus at 20°C, there was 25.9 percent survival in light and 20.6 percent in dark while the respective corresponding viable conidia at 28°C were 17*7 and 15*9 percent; there was an indication that these levels of survival were reached faster in light. For example, at 20°C while viability of 84.7 percent after 10 days fell to 39.0 percent in 5 days, it fell from 81.9 to 5 6 .9 percent in dark within the same period. The great drop in viability at 28°C occurred earlier and the implication of both the temperature and the higher light intensity in this instance would be discussed later. The conidia evidently survived better at the lower tempera­ ture and greater proportion of the conidia was conserved at all sampling times at 20°C. Viability was greatly reduced between 5 and 10 days at 28°C to less than 32 percent, whilst more than 80 percent of conidia stored at 20°C survived after 10 days. University of Ghana http://ugspace.ug.edu.gh - 76. - G-. Longevity of Conidia of T. fructigena stored in water Most observr-tions of longer survival at high humidities recorded in the literature do not contain any information on survival of the spores in liquid water. Physical conditions imposed by liquid water are very different from those of humid air. The longevity of T. fructigena conidia in water could be conveniently tested for in these studies since rather very low germination takes con idiQ place in water and large quantities of ungerminated^would be available for subsequent germination tests. Aqueous spore sus­ pensions in petri dishes were stored in the dark at 25°C. Four dishes were withdra?/n after specific intervals as indicated in Table 13 and the ungerminated conidia sown on petri plates containing PD/i, after germination percentage in the distilled water had been assessed. The few germinated conidia usually floated on the water surface, while the ungcrminated conidia sedimcnted at the bottom of the dish an_ were easily sucked up with a pipette. This spore suspension was then added to an equal volume of double strength of Potato Dextrose Broth (400g. potato, 40g. dextrose, 1000 ml. distilled water) and incubated at 25°C, for 12 hours. On mixture University of Ghana http://ugspace.ug.edu.gh - 77. - the Potato Dextrose Broth was diluted to normal concentration. The percentage germination obtained in the nutrient medium at each sampling time is shown in Table 13. The results indicate that T. fructigena conidia were well conserved in water. Longevity in water was better than at 10($> R.H. (Table 11). Percentage survival after 20 days was 80.9 and 29.7 percent in water and at 100^ o R.H., respectively, (Tables 11 and 13). University of Ghana http://ugspace.ug.edu.gh - 78. - TABLE 13 Longevity of Conidia of T. fructigena stored in Distilled Water at 25°C for 20 Days Germination in Distilled Germination in Potato water during storage Dextrose Broth after Period storage of storage Total Percentage Total Percentage in number Germination number Germination Days of of conidia conidia observed observed 1 1472 0.2 1492 97.1 2 1477 0.5 1493 96.1 3 1483 0.3 1518 92.3 C 1564 0.1 1561 96.0 9 1578 0.2 1576 95-1 12 1513 0.2 1584 90.9 13 1576 0.1 1547 8 2 .7 20 1582 0.0 1609 80.9 University of Ghana http://ugspace.ug.edu.gh H, Effect of loss of water, by Plasmolysis, on Germination Capacity of Conidia of T. fructigena The proportion of spores that collapsed at any relative humidity was closely related to the percentage of conidia that died (Chapter E). The percentage of spores which had collapsed could thus be used as a criterion of the percentage viability of any batch of spores. Was death due to mere loss of water from the cytoplasm or to an effect on the spore membrane or to both? To find whether egress of water from the spore - under conditions that did not permit wall collapse - would kill the conidia at the same rate as those exposed to the atmosphere, the conidia were placed in solutions of commonly used plasmolysing agents, Potassium nitrate, sodium chloride and sucrose for 60 minutes and then sown on petri plates of PHI for 12 hours, to determine their germination capacity. The spore suspensions of plasmolysing fluids v/ere kept in test tubes and the spores were heavily deposited at the bottom of the tubes at the end of one hour. The supernatant fluid was poured off and the spore deposits, with very little of the plasmolysing fluid seeded onto the plates. The plasmolysing fluid was not washed off as it was not known how damaging the University of Ghana http://ugspace.ug.edu.gh sudden movement of via ter into the plasmolysed spores might be. The results are shown in Tables 14 to 16. No germination occurred in any of the plasmolysing solutions and none of the spores collapsed. Percentage survival in all solutions remained above 77 percent even after storage for 60 minutes at osmotic pressure of 29.9 atmospheres (Tables 14, 15 and 16), The high germination obtained indicated that the method followed was reasonable. The little quantity of plasmolysing fluid that was taken up with the unwashed conidia did not obviate the effects of the nutrients of the PDA. University of Ghana http://ugspace.ug.edu.gh TABLE 14 Germination of Conidia of T. fructigena plasmolysed in Potassium Nitrate Solution prior to Incubation on PDA at 25°C for 12 hours Time kept in Plasmo­ lysing solution (Minutes) Molarity (M)* Shrinkage of conidia in Plasmolysing Fluid Total Percentage number Collapse of conidia observed Germination on PDA after Plasmolysis Total Percen- number tage of Germina- conidia tion observed 0 , 1 342 0 .0 376 93.6 20 0 .2 387 0 .0 368 77.4 0.3 356 0 .0 398 8 1 . 2 0.7 370 0 .0 413 79.0 0 .1 390 0 .0 404 92.6 40 0.2 410 0.0 362 91.7 0.3 375 0.0 382 94.0 0.7 386 0.0 356 94.1 0.1 350 0.0 367 93.7 60 0.2 392 0.0 359 95.0 0.3 CD 0.0 352 92.3 0.7 372 0.0 393 89.1 * 0.1H = 4.22 a ta s * .; 0.2M = 8 .04 a tm ® ., 0.3M - 11.2 atiJBi.; 0.7M = 24.6 atmcs. University of Ghana http://ugspace.ug.edu.gh TABLE 15 Germination of Conidia of T. fructigena plasmolysed in Sodium o chloride solution prior to Incubation on PDA at 25 C for 12 Hours. Time kept in Plasmo­ lysing solution (Minutes) Molarity ' (M)* Shrinkage of Conidia in Plasmolysing fluid Total Percentage number Collapse of conidia observed Germination on PDA after Plasmolysis Total Percentage number Germination of conidia observed 0.1 384 0.0 357 96.1 0.2 402 0.0 349 90.5 20 0.3 390 0.0 375 84.8 0.7 373 0.0 372 77.5 0.1 370 0.0 367 96.5 0.2 364 0.0 409 88.8 40 0.3 386 0.0 388 77.3 0.7 399 0.0 376 8 5 .1 0.1 382 0.0 368 93.8 60 0.2 357 0.0 349 96.3 0.3 377 0.0 350 97.7 0.7 396 0.0 396 82.6 * 0.1M = 4.45 atm®.; 0.2M = 8.73 atmta.; 0.3M = 1 3 . 0 atmes.; .0.7M = 29.9 atmB. University of Ghana http://ugspace.ug.edu.gh TABLE 16 Germination of Conidia of T. fructi^ena plasmolysed in Sucrose Solution prior to Incubation on PDA at 25°C for 12 Hours. - 83. - Time kept in Plasmo- lysing solution (Minutes) Molarity 00* Shrinkage of Conidia in Plasmolysing fluid Total Percentage number Collapse of conidia observed Germination on PDil after Plasmolysis Total Percentage number Germination of conidia observed 0.2 375 0.0 344 94.8 0.3 384 0.0 348 96.0 20 0.5 349 0.0 332 95.5 1.0 39» 0.0 339 94.7 0.2 347 0.0 354 98.6 0.3 388 0.0 361 96.1 40 0.5 365 0.0 358 88.0 1.0 372 0.0 371 96.0 0.2 394 0.0 374 94.1 0.3 403 0.0 358 98.9 40 0.5 386 0.0 354 98.0 1.0 354 0.0 378 93.9 * 0.2M = 4.96 atmfeg.; 0.3M = 7.49 atmeai.; 0.5M = 12.4 strata.; 1.0M = 2 5 .0 atm££3. University of Ghana http://ugspace.ug.edu.gh I. Further Experiments on the Effect of Loss of Water by Plasmolysis on C-ermination Capacity of Conidia of T. fructigena The previous experiment showed that T. fructigena conidia plasmolysed in 0.7 H solutions of Potassium nitrate and Sodium chloride and in 1.0 M solution of Sucrose, were hardly affected and conidia o'f all treatments subsequently germinated very well on PDii. The maximum time of immersion was 60 minutes which was long enough to induce plasmolysis. It is possible that the spores have sufficiently high osmotic pressure to prevent excessive plasmolysis. Instances of high osmotic pressures have been recorded in the literature. Osmotic pressures of 63 atmospheres for conidia of Brysiphe graminis and 68 atmospheres for Erysiphe polygoni had been reported by Brodie (1945)* The experiment below was designed to investigate the effect of higher concentrations of sucrose solution only, on the viability of the conidia. The procedure of the previous experiment (Chapter H) was followed, using this time 1.5 and 2.0 M. sucrose solutions. The conidia were transferred to PDA plates after 60 minutes immersion in the plasmolysing solutions and incubated at 25°C for 12 hours, after which percentage germination was assessed. The results are presented in Table 1 7 . University of Ghana http://ugspace.ug.edu.gh TjlBLE 17 Germination of Conidia of T. fructi^ena Plasmolysed in Sucrose Solutions prior to Incubation on PDil at 25 C for 12 Hours. Shrinkage of Conidia Germination on PDA in Plasmolysing after Plasmolysis Fluid Total Percentage Total Percentage number Collapse number Germination of of conidia conidia observed observed 20 354 0.0 363 93.4 1.5 40 339 0.0 378 89.9 60 382 0.0 411 90.5 20 397 0.0 388 90.7 2.0 40 375 0.0 409 87.0 60 407 0.0 419 85.9 Molarity 00 * Time kept in Plasmo­ lysing Solution (Minutes) 1.5M = 37*5 atmos.; 2.0M = 50.0 atmos. University of Ghana http://ugspace.ug.edu.gh The results show that 60 minutes plasmolysis at the higher concentrations of sucrose was not more damaging than the lower concentrations of the previous experiment. As high as 90.5 and 85.9 percent viability were recorded for spores plasmolysed for 60 minutes in 1.5 and 2.0 M sucrose solutions. In the preceding experiment and in the present one, the conidia were found to have been plasmolysed to varying degrees, was T h i s 14^ 30 re-examined in the next experiment. University of Ghana http://ugspace.ug.edu.gh J. longevity of Conidia of T. fructigena stored in Sucrose solutions Cursory exojnination of conidia in the previous experi­ ment showed that they were plasmolysed in the sucrose solutions. Immersion for only one hour was, evidently, too brief to effect serious disruption in the spores judging by the high percentage survival of all the treatments (Tables 14 to 17). Although plasmolysed spores survived far better than conidia that lost water at such high relative humidity as 90fo R.H. (Table 9), it was presumed that they would live - because of their apparent sensitivity to loss of water - for far shorter periods than conidia stored in water, in the unplasmolysed state. This hypothesis was tested by keeping the conidia in both 1.5 M and 2.0 M sucrose solutions for 72 hours. Conidia in distilled water served as control. Table 18 indicates the proportion of conidia that survived plasmolysis in the sucrose solutions after 6, 12, 2 4, 48 and 72 hours. Results in Table 18 show that again no conidia collapsed. Lengthening the period of spores in plasmolysing solutions of 1.5 M and 2.0 M sucrose shortened their longevity. Survival after 72 hours in 1.5 M and 2.0 M sucrose solutions was, respectively, 34.1 and 32.7 percent, whilst a corresponding storage in distilled water (control after 72 hours) showed 94.7 percent viability. - 87. - University of Ghana http://ugspace.ug.edu.gh Longevity of Conidia of T. fructigena stored in 1.5 and 2.0M Sucrose Solutions for 72 Hours. TABLE 18 Molarity (M) Time kept in Plasmo­ lysing solution (Hours) State of Total number of conidia observed Conidia in Solution Percent­ age Plasmo- lysed Sucrose Percent­ age Collapse Percentage on PDA after Plasmolysis Total Percentage number Germination of conidia observed 6 391 100.0 0.0 420 88.8 12 398 100.0 0.0 400 75.8 1.5 24 388 100.0 0.0 397 69.5 48 378 100.0 0.0 385 56.6 72 395 100.0 0.0 414 34.1 6 381 100.0 0.0 419 89-7 12 378 100.0 0.0 426 80.6 2.0 24 377 100.0 0.0 392 87.5 48 371 100.0 0.0 391 63.4 Distilled 72 400 100.0 0.0 410 32.7 H2° 72 - - - 454 94-7 University of Ghana http://ugspace.ug.edu.gh Every conidium observed after storage in the sucrose solutions was found to have plasmolysed. The spores were, however, not plasmolysed to the same extent as demonstrated by Plates 8 and 9. It is not possible from observations here to relate degree of plasmolysis to viability. It could be conjectured that death would occur when a certain stage of plasmolysis had been exceeded. University of Ghana http://ugspace.ug.edu.gh - 90. - Plate 8. Photomicrograph of plasmolysed unstained conidia of T. fructigena after 12 hours incubation in 1.5M Sucrose solution. Note the varying degrees of plasmolysis. (X 200) University of Ghana http://ugspace.ug.edu.gh - 91. - Plate 9. Photomicrograph of plasmolysed unstained conidia T. fructigena after 12 hours incubation in 2.0M Sucrose solution. Note the varying degrees of plasmolysis. (X 200) University of Ghana http://ugspace.ug.edu.gh - 92. - K. Possible Effects of Storage in Water on Metabolism of T. fructigena conidia 'tfhen conidia of T. fructigena were stored at 100 ^R.H. and in water, a high percentage of the conidia survived for more than 10 days and germinated very well on PDA (Tables 11 and 13). It has generally been presumed that metabolic activities of fungal spores would proceed at a high rate under such conditions and the effects would be shown in two ways. Either metabolic activities under high humidity conditions would better 'prepare' the spore for gemination and so hasten gemination when favourable conditions are provided or the attendant accumulation of metabolites within the spore would depress metabolic activity and delay germination. These hypotheses could be examined in various ways. Often the length of the germ tubes is an indication of time of their production. The extensively branch­ ing nature of T. fructigena germ tubes did not permit the use of this criterion. One other method would be an estimation of the rate of germination of the conidia stored for varying periods, by measuring percentage germination at closely-spaced intervals. Such studies were carried out on conidia stored at 100/£ R.H, and in water and at 25°C. The maximum storage time was 10 days when more than 90 per ccnt of the conidia would be alive (Tables 11 and 13). The conidia were, as usual, germinated on PDA for 12 hours at 25°C after storage. The results arc presented in Tables 19 and 20 and graphically represented in Pig.8. University of Ghana http://ugspace.ug.edu.gh - 93. - TABLE 19 Germination of T. fructigena conidia on P M in 12 hours after storage in dark at lOOfo R.H. and at 25°C. Period of Period of Total Percentage storage at incubation number Germination 10C$> R.H. in on P M in of Days Hours conidia observed 2 381 7.3 4 396 16.2 6 393 30.3 . 2 8 348 55-5 1 0 . 385 7 3 .0 12 390 89 .7 2 356 8 .4 4 384 22.1 6 392 38.3 b- 8 343 6 2 .1 10 386 7 2 .3 12 386 90 .2 University of Ghana http://ugspace.ug.edu.gh - 94. - TABLE 19 (cont'd.) Germination of T., fructigena conidia on PDA in 12 hours after storage in dark at 100$ R.H.. and at 25°C. Period of storage at 1t0$ R.H. in Days Period of Incubation on PDA in Hours Total number of conidia observed Percentage Germination 2 381 2 3 .6 4 350 41.1 6 379 58.0 6 8 405 7 0 .6 10 387 78.6 12 372 86.8 2 385 9.9 4 354 20.3 6 356 29 .8 8 8 367 43.9 10 410 65.4 12 400 75-8 University of Ghana http://ugspace.ug.edu.gh - 95. - Germination of T. fructigena conidia on PD^ l in 12 hours after storage in dark at 10C% R.H. and at 25°C. TABLE 19 ( c o n t 'd . ) Period of storage at 10V/o R.H in Days Period of Incubation on PDii in Hours Total number of conidia observed Percentage Germination 2 360 12.5 4 353 24.1 6 388 30.7 10 8 370 45-7 10 348 59.4 12 391 73-4 University of Ghana http://ugspace.ug.edu.gh - 96. - TABLE 20 Germination of T. fructigena conidia on PDA in 12 hours after storage in dark and in water at 25°C. Period of Period of Total Percentage storage in Incubation number Germination Water in on PDA in of Days Hours conidia observed 2 336 43.5 4 358 91. 1 6 349 95.7 2 8 406 96.8 1* 371 94.9 12 390 95-4 2 357 1 9 . 0 4 373 94.4 6 379 97.4 t 8 372 96.0 10 393 95.7 12 370 96 .5 University of Ghana http://ugspace.ug.edu.gh - 97. - Germination of T. fructigena conidia on PDA in 12 hours after storage in dark and in water at 25°C. TABLE 20 ( c o n t 'd . ) Period of storage in water in Days Period of Incubation on PDii in Hours Total number of conidia observed Percentage Germination 2 3 64 23.4 4 416 83.9 6 364 95.3 6 8 394 94.7 1f 385 96.6 12 377 93-9 2 382 21.7 4 340 72.1 6 369 91.1 8 8 366 94.0 10 384 92.4 12 382 93.5 University of Ghana http://ugspace.ug.edu.gh Germination of T. fructigena conidia on PDA in 12 hours after storage in dark and in water at 25°C. TABLE 20 (c o n t 'd . ) Period of Period of Total Percentage storage in Incubation number Germination Water in on PDi; in of Days Hours conidia observed 2 361 3 6 .8 4 407 75-4 6 386 87.3 8 383 92.7 10 380 93.4 12 374 92.0 University of Ghana http://ugspace.ug.edu.gh HOURS OF GERMINATION ON PDA FIG. 8 RATE OF GERMINATION OF T. FRUCTIGENA CONIDIA AFTER STO R A G E AT 100% R.H. AND IN LIQUID WATER FOR 2, 4, 6, 8 ANO 10 DAYS •----- STORED IN LIQUID WATER o-----o STORED AT 100% R.H. University of Ghana http://ugspace.ug.edu.gh Tho results show that rate of germination was faster in conidia stored in water than those kept at 1007$ R.K. For example, maximum percentage germination (91.1 percent) of conidia stored for 2 days in water was attained on PDA in 4 hours (Table 20). Conidia kept for the same period at 100$ R.H. needed as long as 12 hours incubation on PDA to achieve maximum germination (89.7 percent) (Table 19). Conidia stored at 100fc R.H. at different sampling days similarly showed the highest germination at 12 hours. Spores kept in water for 4- days, in contrast, germinated well showing maximum germination after only 4 hours incubation on PDA. Six to ten days' storage in water, however, affected spore vigour. The conidia geminated at a slower rate and maximum germination was observed after 6, 6 and 8 hours' incubation on PDA in conidia stored for 6, 8 and 10 days, respectively. University of Ghana http://ugspace.ug.edu.gh - 101. L. Germination of Conidia of T. fructigena in aqueous extract of native soil Husks of cocoa pods from which the beans have boon extracted are left in heaps on the ground of cocoa farms for very prolonged periods. These heaps naturally form a very good food base for T. fruetigena and, furthermore, form a springboard for soil infection. 'Hie fate of conidia, either washed off from mycelium growing on the pod heaps into the soil, or those formed by mycelium in the soil is of extreme importance in relation to the establishment of the fungus on new crop of pods. If they remain alive in soil, they could be splashed by rain drops onto pods at the lower regions of the trunk, or carried from soil up the trunk by ants. The subject of spore germination has more recently been reviewed by many (Cochrane, 1966; Gottlieb, 1966; Sussman, 1966). Dormancy as defined by Gottlieb (1950) has been qualified by Sussman (1966) as constitutive dormancy, to distinguish it from exogenous dormancy, imposed upon the spore by environmental conditions that inhibit germination. If exogenous dormancy were imposed by soils of cocoa farms on T. fructigena conidia, they could play the role of infection units much better if they do not germinate in soil at least indiscriminately unless in the presence of a food base that would ensure support for the resultant mycelium or until the dormant spore is dispersed onto pods. University of Ghana http://ugspace.ug.edu.gh - 102. - Inhibition of fungal spores by soils has boon described by several investigators (e.g. Chinn, 1953; Dobbs and Hinson, 1953; Jackson, 1958; Stover, 1958> etc.) and the concept of a general widespread fungistasis in soils has been developed. The germination of T. fructi^ona in extracts of soil of a cocoa farm at Aburi, with high mealypod incidence, was examined to find out whether the conidia would bo prevented from germination. Conidial suspensions were separately prepared with non-sterilc extracts of (a) soil (pH 9.9) and (b) partially decomposed leaves (pH 8.2) of the cocoa farm. Mycelial fragments in the suspensions were removed by straining the suspension with sterile muslin cloth. Aliquots of 20 ml of each spore suspension were pipetted into sterile petri-dishes and incubated at 25°C. Percentage germination was assessed after 12 hours. Conidia in distilled water served as control. There was no germination in all three treatments. The minimum examined number of conidia -^oboorvod- during observation of the incubated conidia at the end of the incubation period was more than 740 for each treatment. University of Ghana http://ugspace.ug.edu.gh - 1 0 3 . - M . Cause- of Uxo;renous Soraancy in Conidia of T. fructigena in Soil extract One of the major causes of fungistasis in soil is considered to be the inhibitory action of products of microbial metabolism. Inhibition could, therefore, be removed through elimination of the microorganisms by sterilization (Dobbs and Gash, 1965; Dobbs and Hinson, 1953). Conidia of T. fructi.crena wore, as a sequel to the experiment in the previous chapter, germinated in autoclaved extracts of the par­ tially decomposed loaves and soil for 12 hours at 25°C. There was again no germination in the autoclaved oxtract of partially decomposed leaves (out of 736 observed conidia) and autoclaved soil extract (out of 755 observed conidia). University of Ghana http://ugspace.ug.edu.gh - 1 0 4 . - N, Examination of the Prcscnco of Fungistasis in native soil From the results of the experiment of Chapter L, it could be inferred that fungistasis was present in non-sterile extracts and permitted absolutely no germination. Since there was also no germ­ ination in the sterile extracts (Chapter M), either there was a residual fungistasis which was strong and effective, or nutrients in the extracts were too low to stimulate germination after removal of the fungistatic principle. The results so far do not, however, establish conclusively the presence of fungistasis since the conidia did not also germinate in water. The presence of fungistasis in the extracts could be demonstrated by supplementing extracts of the soils with different concentrations of nutrients. Very high concentrations of nutrients should overshadow the fungistatic effect. On the other hand, at very low nutrient concentrations, fungistasis should exert its influence and allow lower germination in comparison to germination in unamended nutrient of identical concentration. This experiment verified this hypothesis - by using Potato Dextrose Broth of the concentrations N, II/2, n/4> W 3 and N/l 6, N denoting the normal concentration of Potato Dextrose Broth medium, where N = (200g. potato; 20g. dextrose; 1000 ml do— ionised water). Double concentrations of both the media and extracts were prepared in order to achieve the desired concentrations on mixture of equal volumes of the two media. University of Ghana http://ugspace.ug.edu.gh Spore suspensions prepared with the 'mixture' solution were incubated at 25°C for 12 hours. Percentage germination obtained for the variou treatments arc presented in Table 21 and in Pig.9- The results clearly indicate fungistatic effect in extract of partially decomposed leaves. Fungistasis oclipscd, partially, the nutrients of low concentrations, I'l/g and l'l/l6, of the PDB. For example, whilst PDB alone at 1./16 supported 98.6 percent germination, only 17.0 percent of the conidia geminated when the extract was added. The germ tubes were in addition very short. University of Ghana http://ugspace.ug.edu.gh - 106. - TABLE 21 Germination of conidia of T. fructigena in non-sterile Extracts of Soil and Partially Decomposed Leaves supplemented -with Potato Dextrose Broth, at 25°C in 12 hours. PDB amended with Concentration of PDB Total number of conidia observed Percentage Germination N 1481 98.1 K/2 1436 97.8 Soil Extract N/4 N/8 1437 1430 98.2 98.3 N/16 1422 97.9 N 1453 97.3 Extract of Partially Decomposed Leaves K/2 N/4. N/8 1437 1422 1468 97.6 96.8 70-5 N/ 1 6 1512 17.0 K 1456 99.1 Distilled Water K/2 N/4 N/8 1461 1434 1468 98.7 99.0 99.1 N/K 1438 98.6 University of Ghana http://ugspace.ug.edu.gh 100 75 50 25 0 100 75 50 25 0 100 75 50 25 0 9 - 10 7 . - E X T R A C T OF P A R T I A L L Y D E COM PO S ED L E A V E S D I S T I L L E D W A T E R N/ N , N , N , N 16 ' B '4 '2 N O N C E N T R A T I O N O F P O T A T O D E X T R O S E B R O T H S O I L E X T R A C T FFECT OF FUNGISTASIS IN POTATO-DEXTROSE NRICHED EXTRACTS OF SOIL AND P A R T I A L L Y DECOMPOSED LE AVES ON GERMINAT ION OF CON ID IA OF T. FRUCT IGENA AT 25 ° C University of Ghana http://ugspace.ug.edu.gh - 108. 0. Possible Volatile I.ature of the Fungistatic Principle of native soil with the observation of reduced germination at N/8 and N/16 concentrations of the PDB amended with the extract of partially decomposed leaves, a positive evidence of presence of fungistasis had been established.. An investigation was next carried out t« define further the nature of the fungistatic principle of this soil. Recent investigations have shown that the fungistatic principle is volatile (Hora and Baker, 1970; 1972), Such a property thus makes fungistasit. m#re effective, as spores distantly placed could be influenced as well. It was thought desirable to find out whether the inhibitory principle of the &buri soil was volatile. Fig.10 demonstrates the method adopted in this investi­ gation. A small quantity of m*lten PDA (5 nil) was poured into the lid of a sterile petri dish and was spread by gently tilting the lid, so that it set into a thin flat layer in the centre of the lid. A piece of culture of T. f'ructi^ ena in a petri plate was then cut out with a sterile scalpel, lifted with a flamed inoculating needle, and the Oultur. 3urfr.ee gently adpressed to the surface of the agar in the lid. The lid was then placed University of Ghana http://ugspace.ug.edu.gh - 109. - FIG. 10 S E C T I O N A L D I A G R AM I L L U S T RA T I NG CON I D I A OF T . F R U C T I G E N A SOWN ON P DA ON P £ T R I L I D S EXPOSED TO SO I L E X T R A C T . University of Ghana http://ugspace.ug.edu.gh - 110. - back onto the bottom half of the petri dish which had been almost fully filled with lion-sterile soil extract, leaving a space of only 2 mm between the surfaces of the agar and the extract. In this condition, the conidia would be directly under the influence of any volatile matter emanating from the extract. FDA of N/8 and N/16 concentrations only 7/ere used since fungistasis was only detected in the presence of these low nutrient concentrations in the previous experiment (Table 21). When the conidia were examined after incubation at 25* C for 12 hours, germination was similar in all three treatments, viz, petri dishes holding distilled water (control) and extracts of soil and partially decomposed leaves (Table 22). Even on N/16 PDA, the respective percentage germination was 93.5, 92.9 and 93.6 percent in the presence of water, and extracts of soil and partially decom­ posed leaves. The fungistatic principle was seemingly non-volatile. University of Ghana http://ugspace.ug.edu.gh - 1 1 1 . - TABLE 2. Germination of T. fructigena conidia sown on P M and exposed to non-sterile extracts of soil and partially decomposed leaves for 12 hours at 25°C. Extract Concentration of PDA Total number of conidia observed Percentage Germination Loamy N/8 374 93.6 Soil N/16 396 92.9 Partially N/8 387 97.4 Decomposed Leaves N/16 391 93.6 Distilled N/8 396 84.9 Water N/16 387 93.5 University of Ghana http://ugspace.ug.edu.gh - 112 . - P. Survival of Conidia of T. fructigena in Extracts of native soil Extracts of soil of the cocoa farm at Aburi were fungistatic and they imposed exogenous dormancy on conidia of T. fructigena. Jin information of practical importance is the period for which viability of the conidia would be sustained under that condition. There are various possible factors that would prevent long survival. Excessive mineral salts would impose plasmolysis problems. The spores may form substrates for soil bacteria. Lingappa and Lockwood (1961) have shown that spore-surf'ace bacteria subsist on spore-exudate nutrients. The rate at which bacteria would flourish would be closely related to the pH of the soil, and they would most likely operate best at alkaline pH, following the observations of Jackson (1958) which showed that fungistatic effect decreased with increasing acidity- conditions that prevent bacterial action and growth. Some of these problems were considered in the following investigation. Conidial suspensions were prepared with extracts of soil and, partially decomposed leaves adjusted to pH 4 .0 , 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, measured with capillators, with either dilute hydrochloric acid or }.1N sodium hydroxide and stored at 25°C for 15 days, lifter storage, the conidial suspensions were separately seeded onto PDA University of Ghana http://ugspace.ug.edu.gh - 113. - and incubated at 23°C for 12 hours before assessing percentage germination. The results are presented in Table 23 and in Fig.11. More than 50 percent of the spores was still viable after 15 days at some pH levels. The results show that the best pH for survival was pH 8.0, where 69.6 percent of the conidia in the soil extract survived 10 days' storage and 3 1 . 0 percent was alive after 15 days. Longevity in the extract of partially decomposed leaves was closely similar; 60.6 and 56 .8 percent survival, respectively, after 10 and 15 days. Survival then declined as the pH moved to both the acidic and the alkaline ends. In both extracts pH 4.0, which was farthest removed from the optimum, was most unsuitable for storage (Fig.11). University of Ghana http://ugspace.ug.edu.gh TABLE 23 Survival of T. fructigona conidia stored for 15 days at 25°C at various pH levels of extracts of Partially decomposed leaves and soil, (Conidia germinated on PDA for 12 hours at 25°C after storage). - 114. - Period of storage in Days pH of Extract -Survival in extracts of: Partially Decomposed Loar Soil leaves Total Percentage Total Percentage number Germination number Gemination of of conidia conidia observed observed 4.0 381 10.0 412 14.1 5.0 389 42.4 415 14.7 6.0 398 43.0 423 40.9 10 7.0 412 53.9 395 44.8 8.0 393 60.6 424 69.6 9.0 433 47.3 411 66.6 10.0 412 50.5 455 67.9 4.0 458 23.4 385 3.1 5.0 414 29.2 419 25.5 6.0 446 48.7 427 39.1 15 7.0 444 54.7 412 49.8 8.0 414 56.8 414 51.0 9.0 440 32.3 453 37.7 10.0 454 30.2 435 42.0 University of Ghana http://ugspace.ug.edu.gh V IA B IL IT Y % V IA B IL IT Y - 1 1 5 . - 8 o r 6 0 40 80 6 0 ■ 4 0 - 8 0 J L _L_ J_______ |_ J _ E X TRACT OF PARTIALLY DECOMPOSE LEAVES SOIL EXTRACT 5 6 7 8 9 10 pH OF STORAGE MEDIUM FIG. II SURVIVAL OF T. FRUCTIGENA CONIDIA INCUBATED FOR 10 AND 1 5 DAYS IN E X T R A C T S OF P A R T I A L L Y DECOMPOSED L E A V E S AND SOIL A D J U S T ED TO VAR IOUS pH L E V E L S . (CONIDIA GERMINATED ON P D A FOR 1 2 HOUR S A F T E R S T O R A G E ) 10 DAYS OF INCUBATION 15 DAYS OF I NCUBATION University of Ghana http://ugspace.ug.edu.gh - 1 1 6 . - Q. Lysis of Conidia of T. fructigena in native soil In most studies on fungistatic effect of soils, attention has mostly been given to the fate of the spore or hyphae. In his review on factors limiting survival and activity of fungi in soil, Brian (1960) explained that heter^lysis could be due to oxygen-lack because of a large microbial population and due also to some anti­ biotics acting as respiratory inhibitors- He cited further examples of bacterial and actinomycete enzymes that can disrupt fungal cell walls or protoplast membranes. If the spore is capable of sufficient metabolism under soil conditions, it is possible that metabolites of a spore and hypha could in turn exert inhibitory influence on micro-organisms close to them and they could, at least, delay their destruction. There is evidence from the present study that T. fructigena conidia could do so. In this experiment, T. fructigena conidia in two states were used, dead and viable spores. To kill the conidia, spore prints were incubated over anhydrous calcium chloride, to desiccate the spores, for 6 hours. The slides bearing the conidia were then buriud in loo.m soil from the cocoa farm at b^uri, and adjusted to 2Q& moisture content. University of Ghana http://ugspace.ug.edu.gh - 117. - Four slides bearing the spore prints were then placed vertically, standing on the longer edge, in a glass bowl (14 cm. diameter and 6 cm. deep) filled with the soil. In order to avoid wiping off the conidia during insertion of the slides, four parallel equiaistantly spaced slits were made with a scalpel into which the slides were put. The soil was then pr.ssed firmly against the slide. state There were four bowls of four slides each for each^ fey^ ^of spore. The bowls were finally covered with polythene sheets held in position with rubber bands to keep the moisture content constant and incubated at 25°C. Two bowls, one containing slides bearing initially dead conidia and the other living conidia, were withdrawn at 24-hour intervals. drop of distilled water was quickly placed on the viable spore print on withdrawal of the slide to prevent desiccation and the conidia examined under the microscope for presence of micro­ organisms and signs of degradation. The conidia were then, as spore suspension, germinated on PDA in petri plates at 25°C for 12 hours after v/hich assessment of percentage germination (percentage survival) was made. University of Ghana http://ugspace.ug.edu.gh - 1 1 8 . - The results presented in Table 24 and 25 clearly show a difference in the extent of visible attack by micro-organisms between originally viable and dead conidia. On the 4th day only 0.9 percent of the 1690 initially viable conidia observed carried a microflora visible under high power of the microscope. Survival after 4 days in the soil was 54-5 percent. Dead conidia, on the other hand, showed heavy incidence of saprophytes. Out of 1814 conidia observed after 4 days' burial in the soil, 85-9 percent had been visibly attacked by saprophytes (plate 10) end 22.1 pcrcent was completely lysed and they could be only identified by their faint outline (Fin.to 11). Such lysis had seemingly been delayed in spore prints of viable conidia as no lysis was detected after 4 days' burial in soil. This impression was confirmed by a supplementary experiment in which the conidia of initially viable spore print were examined under the microscope after withdrawal from the soil without adding the drop of distilled water. This kept the conidia in position and would allow detection of any lysed spores. Those spores were not germinated on PM, as they might have perished after exposure during examination under the microscope. University of Ghana http://ugspace.ug.edu.gh - 119. - TABLE 24 Survival of viable conidia of T. fructigena buried in native soil (2C%> vY.H.C.) on slide for 4 days at 25°C. Period in Conidia with no visible signs of microbial attack Conidia with visible signs of microbial attaok coil (Days) Totalnumber of conidia observed Percentage Germination Total number of conidia observed Number of conidia Germinating 1 1435 60 . 1 17 13 2 1847 61.4 20 14 3 1569 54-7 7 4 4 1690 54.5 16 8 University of Ghana http://ugspace.ug.edu.gh - 120 . - T^BLE 25 Degradation of dead conidia of T. fructigena buried in native soil (2C$> w.H.C.) on slide for 4 days at 25°C. Period in soil (days) Total number of conidia observed Percentage of conidia with visible signs of microbial attack Percentage of conidia lysed 1 2210 29-4 0.1 2 2113 37-6 6-5 3 1 6 36 76.3 1 6 . 8 4 1 8 1 4 85-9 22.1 University of Ghana http://ugspace.ug.edu.gh '■ l i n ' U f - Plate 10. Photomicrograph of Actinomycetes growing on 'dead1 conidia of T. fructigena buried in soil for 4 days. (X 200) University of Ghana http://ugspace.ug.edu.gh - 122. - Plate 11. Photomicrograph showing outline of lysed 'dead' conidia of T. fructigena buried in soil for 4 days. (X 400) University of Ghana http://ugspace.ug.edu.gh - 123. - R. Saprophytic Survival of T. fructigena in soil There are no reports on the occurrence of T. fructigena in soil. The most important reason is that it has not yet been sought. It has been suggested earlier (Chapter L) that the practices of cocoa farmers in West Africa are likely to encourage the invasion of the soil by this fungus. As a sequel to the present findings on the fate of spores of T. fructigena in soil, the ability of the mycelium to live as a saprophyte in soil was studied. For a long time interest in the behaviour of pathogenic fungi in soil has been confined to root-infecting fungi. Garrett (1950) reviewed the inter-relationships between root-disease fungi and the associated soil microflora and he distinguished between specialised root-inhabiting parasites and unspecialised soil- inhabiting parasites. This classification did not include shoot pathogens. Garrett (1970) has recently enumerated all possible modes of survival of root-disease fungi that have been discovered during the numerous pertinent investigations: (1) as competitive saprophytes on dead organic substrates; (2) saprojjhytic survival on dead tissues of a host crop or weeds, infected during•the parasitic phase; University of Ghana http://ugspace.ug.edu.gh - 124. - (3) dormant survival as ’resting1 propagules, e.g. sexually produced oospores and other spores, asexually produced chlamydospores, and multicellular sclerotia; (4) parasitic survival on living roots and other underground parts of used hosts and 'volunteer' susceptible crop plants; (5) parasitic survival on living root systems of plants that show no disease symptoms above ground. At least T. fructigena, a facultative parasite, should be able to use the first two modes of survival. The present work investigated this. The soil used was loam soil from the cocoa farm at Aburi. It was passed through a 2 mm sieve and adjusted to a moisture content of 4 ^ saturation capacity, A maize meal-sand medium of 100 parts sand, 3 parts ground maizemeal and 13 parts water (by weight) was in the meantime inoculated with T. fructigena and the culture allowed to grow over a month. The culture was shaken occasionally to ensure thorough growth of the fungus in the entire medium. At the end of the month, the University of Ghana http://ugspace.ug.edu.gh - 125 . - culture was also adjusted to a moisture content of 45% saturation capacity and then mixed with the unsterilised loam soil in the following proportions, the culture, hereafter referred to as inoculum, coming first in the ratios: 100:0 (inoculum control); 95:5; 90:10; 7 5:2 5; 5 0 :5 0; 25:75; 10:90; 5:95 and 0:100 (soil control). Immediately after the preparation of the mixture, 50 pieces of dried unsterilized cocoa pod husk and coffee pericarp used as baits were stirred into 400 g portions of the inoculum/soil mixtures in Kilner jars and the jars with their contents weighed. The jars prevev\+ecl were covered with perforated aluminium foil sheets which 3^-v~sddcd^ rapid drying of the inoculum/soil mixture but ensured sufficient jays Were. aeration. Throughout the incubation period of 4 weeks,. K weighed on each other day and the amount of water lost was made up with sterile distilled water. The jars were stacked on a wooden platform in the laboratory (26 + 1°C) with legs standing in bowls of oil to avoid invasion by non-soil crawling insects and flying insects were kept off by a cover of nylon mosquito netting. The baits were prepared in the following way: the husk of cacao pods and the pericarp of coffee berries were cut into small uniform pieces, approximately 1 cm Try .2 cm. The cocoa pods were first peeled before the pieces were cut. The pieces were 5 mm thick. University of Ghana http://ugspace.ug.edu.gh - 126 . - The husk and pericarp pieces were separately dried in the sun until they had completely dried to uniform weight. They were not steri­ lized and were suspended in sterile distilled water for 1 hour immediately before they were used. At the end of the 2f-week incubation period, the baits were recovered, washed free of adhering soil and inoculum and surface sterilized by immersing for 1 minute in mercuric chloride of dilution 1:1000. They were then rinsed twice with sterile distilled water containing streptomycin. Each bait was cut into two but kept together and plated out - five (10 half pieces) per petri dish - on P M containing a drop of 1% solution of streptomycin. The plated baits were incubated at 25° C for five days and the number of baits yielding T. fructigena was recorded. Despite the immense growth of saprophytes on the plates it was possible to identify T. fructigena by its distinctly recognisable echinulated conidia and their unique mode of production on the conidiophores, by careful microscopic examination. This was an extremely time consuming but rewarding exercise that required 3 days for a complete examination of each experiment. The results are presented in Table 26. University of Ghana http://ugspace.ug.edu.gh - 127. - TABLE 26 Colonization of unsterilized Cocoa pod husk and Coffee berries in soil by T. fructigena. (Percentage colonized: based on number of baits (out of a total of 50) bearing mycelium of T. fructigena, identified by the presence of the conidia, after incubation on P M for 5 days at 25°C). Inoculum » ) Percentage Coffee fruits colonized Percentage Cocoa pod husk colonized 100 58 70 95 14 30 90 10 12 75 6 0 50 6 2 25 4 0 10 0 0 5 0 0 0 0 0 University of Ghana http://ugspace.ug.edu.gh - 128 . - The results indicate that T. fructigena has limited competitive saprophytic ability. In cocoa pod husk, saprophytic survival of T. fructigena fell rapidly from 70 percent survival in 10C$> inoculum to 2 percent survival in $0% inoculum. The decline in percentage survival conversely followed an increase in unsteri­ lized loam soil. Similar trend - from 58 percent survival in 10Cf/o inoculum to 6 percent survival in 50^ inoculum, for the pericarp of coffee was observed. Curiously, T. fructigena survived better in cocoa husk at higher percentage inoculum than in coffee pericarp, but this was reversed with the increase in unsterilized soil. Ji. second method of assessment was attempted using only cocoa pod husk pieces, anticipating a less exhaustive method of assessment than the 'Agar plate method'. This was the 'cocoa pod infection method'. Infection through baits containing the fungus would be expected to produce encrusted mass of conidia of T. fructigena on the pod surface (see Plate 1). The baits from the inoculum of soil mixtures at the end of the 4th week, were placed in wounds made in the pericarp of mature green cocoa pods, after they had been surface sterilized with mercuric chloride. The cocoa pods were washed and surface sterilized with 5$ University of Ghana http://ugspace.ug.edu.gh - 129. - sodium hypochloritc. Triangular wounds, each side measuring 2 cm, and 5 nua deep were made with a flamed scalpel. Tho piece of husk removed was placed back after the bait had been inserted. The thickness of the bait, however, lifted the 'lid' and extensively exposed the cut surface. The inoculated pods were finally placed on moist cotton wool inside cellophane bags on the laboratory bench. There was no infection of any of the cocoa pods. It is probable that nutrients from the husk did not diffuse sufficiently well into the bait to encourage the growth of mycelium of T. fructigena present in the bait. Besides this set-back, the large number of pods needed for this method of assessment would be a deterrent. In this experi­ ment 450 pods, kindly provided by the Agronomist of the Cocoa Research Institute, Tafo, were used. University of Ghana http://ugspace.ug.edu.gh - 130. - S. Further Experiments cn the Saprophytic Colonization of Cocoa pod husk buri'-d in soil by T. fructigena The considerable difference between the number of buried cocoa pod husk pieces colonised in the medium of 100 percent inoculum and that of 95 percent inoculum in the previous chapter (Table 26) showed that T. fructigena was not very successful in the colonization of cocoa pod husk in competition with microflora of the soil. Colonization by a test fungus can be improved by various means. Naturally by invading the tissues first, the fungus would establish itself well before the entry of other mioroflora. Butler (1953) demonstrated this in a very simple way. He coated wheat straws with the inoculum of Ophiobolus graminis before burying them in a culture/soil mixture and compared colonization with straws coated with soil and those not treated at all before burial. There was increased colonization by 0. graminis of the straws coated initially with inoculum. There was low colonization of straws coated with soil, which may conversely be attributed to the comparable advantage thereby given to other soil micro-organisms. Butler's experiment was repeated in this investigation. The soil was prepared and maize meal-sand culture of T. fructigena raised according to the procedure in Chapter R. Cocoa University of Ghana http://ugspace.ug.edu.gh pod husk pieces were used as baits and buried in inoculun/soil mixtures prepared to provide inoculum concentrations of 93$, 90fo ~l% and 23^ . Three sets of the experiment were set up. Maize meal-sand culture was pasted onto the baits of one set, while baits of the second set were plastered with moist soil. The third set was a repetition of the previous experiment. The results are presented in Table 27 and Fig.12. The results clearly indicate that the position of T. fructigena in relation to its competitors is an important factor. In the 95*'5 inoculum/soil mixture, 40 percent coloniza­ tion was recorded for husk pieces coated initially with inoculum while only 24 percent colonization was recorded for husk pieces coated initially with unsterilized soil. This advantage still persisted even with increased soil content in the mixture with 7^& inoculum. Cocoa pod husk pieces buried without any treatment showed better colonization than the soil-coated husk pieces and inferior coloni­ zation to inoculum-coated husk pieces. University of Ghana http://ugspace.ug.edu.gh - 132- - TABLE 27 Colonization of unsterilized Cocoa pod husk in soil by T. fructigena placed at different positions from the baits in relation to soil microflora. (Percentage colonized: based on number of baits (out of a total of 50) bearing mycelium of T. fructigena, identified by the presence of the conidia, after incubation on PDA for 5 days at 25°G). Percentage of husk colonized Inoculum « Husk coated with inoculum Husk coated with unsterilized soil Husk placed in inoculum/soil mixture 95 40 24 30 90 24 1? 1 6 75 10 0 6 25 0 0 0 University of Ghana http://ugspace.ug.edu.gh PE R C EN TA G E SU R VI VA L OF T. F R U C T IG E N A - 1 3 3 . - PERCENTAGE INOCULUM FIG. 12 CO LON IS A T ION OF B A I T (CACAO POD HUSK ) BY T. F R U C T IG E N A P LACED A T VAR IOUS P O S I T I O N S FROM T H E B A I T S • ---------- • B A I T S C O A T E D W I T H IN OCU LUM A ----------- A B A I T S C O A T E D W I T H SO IL o ----------o N O N - C O A T E D B A I T S University of Ghana http://ugspace.ug.edu.gh - 134. - T. Saprophytic Colonization of Cacao pod husk of increased nutrient content in soil by T. fructigena It is generally true that the population level of a particular fungus in natural soil, and also its inoculum dosage in an experiment, is one of the most important factors determining the share of a substrate that will be obtained by that fungal species in competition with others. But it is not the only factor. The success of a fungus in competitive colonization will also be affected by such important factors as nutrients available in the substrate and environmental conditions. These will affect its rate of growth on the substrate, and similarly that of its competitors. Nutrients have been shown to be one of the most important soil factors that would influence saprophytic colonization. Garrett (1938) provided the earliest evidence in his work on Ophiobolus graminis« 0. graminis was found to die out early from the rigid and little-decomposed wheat straws burled in nitrogen- poor soils, whereas it was found to survive in almost 100 percent of straws in nitrogen-rich soils. This finding has been confirmed by Butler (1953; 1959), Chambers and Flentje (19^ 9) and Macer (1961). 0. graminis dies out pre-maturely from carbon starvation in wheat straws buried in nitrogen-poor soil, because it cannot obtain the nitrogen needed for continued mycelial growth and hydrolysis of University of Ghana http://ugspace.ug.edu.gh fresh areas of cellulose within the straw tissue. The effect of nutrients, particularly nitrogen, has not been found to be uniform. Butler (1959) discovered that excess soil nitrogen actually reduced longevity of Cochlioboius sativus in wheat straw, while Curvularia ramosa appeared to be indifferent to the level of soil fertility and nitrogen content. These contrasting effects of nutrients have been variously interpreted and the various views have been mentioned in the discussion of this thesis. The response of T. fructigena to added nutrients has been investigated here, as an indication of how it might be affected in soils with different nutrient content. This was an extensive investigation that covered a period of 3 months, consisting of three experiments, set up in series. These were: (a) 4lpril 1973 “ May 1973: the effects of three nitrogen compounds, ammonium tartrate, peptone and sodium nitrate were examined and the compound encouraging best survival sodium nitrate - was used in subsequent experiments. University of Ghana http://ugspace.ug.edu.gh (b) May 1973 - June 1973: the effects of various concentrations of Dextrose and sodium nitrate were examined and the concentrations for best survival selected for the third experiment. (c) June 1973 - July 1973: the effects of a combination of dextrose and sodium nitrate was compared with those of the compounds when present separately. Cocoa pod husk cut out into pieces and dried were soaked in the various solutions for 2 hours and then stirred into the various maize-sand culture/soil mixtures shown in Tables 28 to 31 and incubated at room temperature, 26°C, for 4 weeks, in each case. The husk pieces were then plated on PD& in petri plates for 5 days at 25°C, after they had been surface sterilized with mercuric chloride. The results are presented in Tables 28 to 31 and Figs. 13 to 16. Of the three nitrogen compounds, sodium nitrate encouraged best survival (To,bio 28). Approximately 30 percent of the buried cocoa pod husk soaked in O.fo (30 percent colonization) and \ (36 percent colonization) sodium nitrate solutions contained - 13<- - University of Ghana http://ugspace.ug.edu.gh - 137. - TABLE 28 Colonization of unsterilized Cocoa pod husk, pre-treated with various Nitrogen Compounds, in soil by T. fructigena. (Percentage colonization: based on number of baits (out of a total of 30) bearing mycelium of T. fructigena, identified by the presence of the conidia, after incubation on PDA for 3 days at 25°C). Nitrogen Compound Concentration $(wA) Percentage colonization 0.3 30 Ammonium tartrate 1 . 0 44 2 .0 20 0.5 20 Peptone 1 . 0 6 2 . 0 16 0.5 50 Sodium nitrate 1 . 0 40 1.5 56 University of Ghana http://ugspace.ug.edu.gh - 138. - TABLE 29 Colonization of unsterilized Cocoa pod husk, pre-treated with various concentrations of sodium nitrate, in soil by T. fructigena. (Percentage colonized: based on number of baits (out of a total of 50)’ bearing mycelium of T. fructigena, identified by the presence of the conidia, after incubation on PELi. for 5 days at 25°C). Concentration SS(wA) Percentage Inoculum Percentage Colonized 100 50 0 . 5 95 50 90 30 75 40 100 40 1 . 0 95 36 90 40 75 30 100 56 1 . 5 95 9* 56 75 44 University of Ghana http://ugspace.ug.edu.gh - 139. - TABLE 30 Colonization of unsterilized Cocoa pod husk, pre-treated with various concentrations of Dextrose, in soil by T. fructigena. (Percentage colonized: based on number of baits (out of a total of 50).bearing mycelium of T. fructigena, identified by the presence of the conidia, after incubation on PDA for 5 days at 25°C). Concentration Percentage Percentage %(, W/v) Inoculum Colonized 100 16 0 .5 95 16 90 20 75 4 100 20 1 .0 95 10 90 20 75 • 100 10 2 .0 95 10 90 1 4 75 • University of Ghana http://ugspace.ug.edu.gh - 140. - TABLE .31 Colonization of unsterilized Cocoa pod husk, pre-treated with Dextrose, sodium nitrate and Dextrose-Sodium nitrate mixture, in soil by T. fructigena. (Percentage colonized: based on number of baits (out of a total of 50) bearing mycelium of T. fructigena, identified by the presence of the conidia, after incubation on PD<& for 5 days at 25°c). Percentage colonization of husk treated with Inoculum 1,C%(w/T) 1.^(wA) 1 .($(w/V) ($0) Water Dextrose Sodium nitrate Dextrose + i .5%(wA ) Sodium nitrate 10 0 66 2* 60 90 95 43 2C 60 1 * 0 90 16 2# 50 86 75 4 4 50 50 University of Ghana http://ugspace.ug.edu.gh — 1 4 1 . — FIG. 13 COLON ISAT ION OF BAITS (CACAO POD HUSK ) AMENDED WITH SOLUT IONS OF VAR IOUS NITROGEN C OMPOUND S University of Ghana http://ugspace.ug.edu.gh S U R V IV A L 1 4 2 . F IG. 14 C O LON I S A T IO N OF B A I T S (CACAO POD HUSK ) AMENDED W IT H VAR IOUS CONCENTRAT IONS OF SODIUM N I T R A T E BY T. F R U C T I G E N A University of Ghana http://ugspace.ug.edu.gh S U R V IV A L - 143 . - FIG. 15 COLONI SAT ION OF BA I TS (CACAO POD HUSK) AMENDED WITH VARIOUS CONCENTRATIONS OF D E X T RO SE BY T. F R U C T I G E N A University of Ghana http://ugspace.ug.edu.gh PE R C EN TA G E S U R V IV A L OF T. FR U C TI G E N A - 144. - PERCENTAGE INOCULUM FIG. 16 COLONISATION OF BAITS (CACAO POD HUSK) AMENDED WITH DEXTROSE AND SODIUM NITRATE BY T. FRUCTIGENA (Data f rom Tables 29 , 3 0 and 31 ) • ----------- • l ' 0% DEXTROSE + l '5% SODIUM NITRATE O---------- O l -5% SODIUM NITRATE, A A l ' 0%DEXTR0SE University of Ghana http://ugspace.ug.edu.gh - 145. - T. fructigena after 4 weeks. Saprophytic survival was slightly less in buried pod husk containing ammonium tartrate, ranging between 20 percent colonization at 2.Ofo ammonium tartrate to 44 percent colonization at 1,Q$ ammonium tartrate. T. fructigena did not persist as long in buried cocoa pod husk with peptone. The highest survival was 20 percent, recorded in husk with 0.5% peptone (Fig.13). Untreated buried cocoa pod husk had earlier shown rapidly declining percentage colonization with decreasing quanti­ ties of inoculum (Tables 26 and 31)« In soil containing 90 percent inoculum, only 1 2 percent of the buried husk contained T. fructigena after 4 weeks. Colonization was vastly improved by the addition of sodium nitrate and for the same level of inoculum dosage, sodium nitrate of 0.5 - 1.5% concentration sustained a colonization of 30 “ 56 percent. (Tables 29 and 31; Fig.14) T. fructigena did not persist extensively in cocoa pod husk with dextrose. Even in 100 percent inoculum, the best survival after 4 weeks was 20 percent at 1.C^ dextrose (Table 30; Fig.15). University of Ghana http://ugspace.ug.edu.gh Table 31 clearly demonstrates that a combination of dextrose (1.C$ 7./V) and sodium nitratj (1.5$ W/V) highly increased percentage colonization of buried cacao pod husk by T. fructigena. The fungus persisted in 50 percent of the cacao pod husk buried in soil containing only 75 percent inoculum, while approximately maximum colonization occurred at 95 and 1§0 percent inoculum (Table 31; Fig.16). University of Ghana http://ugspace.ug.edu.gh - 147. - GENERAL DISCUSSION The most important crop in Ghana is cocoa and any malady of this crop is of immense concern to us. T. fructigena is a pod pathogen of cocoa causing mealypod disease (see Plates 1 and 2). The extent of damage caused "fcy the fungus is at present considered insignificant. Judging from the ease with which infection was obtained during the present studies by artificial inoculation and the frequent appearance of the disease on a particular farm at 4buri, it is likely tho.t the disease may reach economic levels in cocoa heavily infested with rodents which will both create wounds for the entry of the pathogen and carry the fungus from pod to pod. The object of the present investigation was to define factors that influence survival of conidia of T. fructigena and examine other aspects of survival of the fungus as a contribution to our knowledge on this fungus of which very little is known. There are other reasons for undertaking this investigation. Its importance in Ghana, at least, is misjudged, and it is supposed to be of uneconomical importance because it causes a very small annual loss of cocoa of only one percent. It is, however, not a pathogen of cocoa alone. It o,ttacks fruits of the important crops, banana, University of Ghana http://ugspace.ug.edu.gh (Brun, 1955; Meredith, 1960; Simmonds, 19&0), Coffee (Bunting, 1924; Hore'fcat'i, 1937; Roger, et al-, 1937) and Avocado pear (Bunting, 1924)* Spread from one crop to the other is very readily encouraged under the common agricultural practice of mixed farming in West Africa, as isolate of one crop infects the other host plants. Tabor and Bunting (1923) reported that experimental cross inoculations from coffee to cocoa and vice versa gave typical symptoms of the disease in each case. T. fructigena has, besides, been found as a parasite of wild forest trees which could act as disease reservoir* Turner and Lovi (19 6 2) found T. fructigena on fruits of Mimusops elengi and Mimusops cummorsonii in Ghana. Finally it has become a serious parasite elsewhere. It is considered serious enough as a pathogen of banana to merit control measures. The disease becomes very severe from June to December in Cameroons necessitating control spraying on a 2-day cycle (Ann. Rept. of the Cameroons Development Corporation, 1958). A thorough knowledge of the biology of the fungus would be invaluable in the design of any successful control measure, especially if the attack is to be directed at the most vulnerable phase of the pathogen. University of Ghana http://ugspace.ug.edu.gh The ccnidia produced in such large masses on the infected pod would unquestionably be of infinite importance in the epidemiology of mealypod disease of cocoa. Very great differences have been found in the survival potential of different kinds of fungal spores. The viability of all spores decreases with time, and.rate of loss of vigour is dependent on the inherent characteristics of the spore and upon environmental conditions, especially, temperature, humidity and light (.Ainsworth and Sussman, 1968; Cochrane, 1958). Pertinent information from the literature has already been mentioned in Chapter E. High temperature shortens viability while 3UT*u->vn 1 i=a= p&r/nits long surviuAl- e-:rrelg/tod -ypith -Iow temperature^ It is probable that moderately high temperatures increase the respiration rate of the quiescent spore resulting in the accumulation of deleterious metabolites and early death. Very high temperatures would obviously kill the spore by denaturing its proteins. The period of viability may be longer in dark than in light in some fungus 3pores (Clerk and Madelin, 1965; Dillon-Weston and Hainan, 1931; Markett, 1953; Tinline et al., 1960; Wolf, 1934). University of Ghana http://ugspace.ug.edu.gh - 1 5 0 . - Dillon-Weston and Hainan (1932) showed that the most active portion of the visible spectrum was blue light. It is generally believed that spore pigment protects the spores against radiation. Dillon- Weston and Hainan (1931, 1932) showed that white and orange spores of Puccinia graminis tritici are more easily killed by ultraviolet light than are grey and red spores. The survival value of pigmentation is generally recognised. Ingold (1953) on coprophilous fungi remarked: "There is evidence that light has an injurious effect on fungal spores, and it is, therefore, of interest to notice in these three fungi that in the spore-masses discharged on to the grass the protoplasm is shaded by the pigment of the spore-wall in Dasyobolus and Sordaria and by the black part of the sporangia1 wall in Pilobolus which effectively covers up the discharged mass of almost colourless spores cemented to a blade of grass". He continued further on, "It is, however, interesting to notice that coprophilous agarics have black (Coprinus. Anelloria, Panaeolus) or dark-brown (stropharia semiglobata) spores, so that again the protoplasm is protected from light during the sojourn of the spores on the grass before it is eaten". University of Ghana http://ugspace.ug.edu.gh Humidity also greatly influences the length of time during which fungal spores remain viable. Quiescent fungal spores show four different types of responses to humidity (see Chapter E). Naturally humidity has different effects on the conidia of the various survival patterns. In common with all observed fungal spores in storage, conidia of T. fructigena survived longer at the lower temperature of 20°C than at 28°C (see Table 12). The influence of temperature is reflected especially by the rate at which the levels of viabi­ lity on the 20th day of storage were achieved. The higher temperature destroyed the spores quicker as demonstrated by a viability of 31 • 7 percent in dark at 28°C in contrast to 81.9 percent viability at 20°C after 10 days. In light, the difference was more accentuated. It is likely that the higher light intensity of 467.5 might have contributed in addition to the temperature effect. It is unlikely that death under these circumstances would be due within periods as short as 10 days to either accumulation of toxic products of increased respiration or to loss of essential reserves. Denaturing of the spores* proteins was more University of Ghana http://ugspace.ug.edu.gh likely to supersede the effect of higher respiration rate, for the conidia seemed to be sensitive to even moderately high temperatures. Supporting evidence would be found in the germinating experiments of Chapters B and D. In contrast to many mesophilic tropical fungi, the conidia barely germinated at 35°C, Only 0.3 percent of the conidia 7/as able to germinate on PDA at this temperature. Also the conidia which do not germinate at 40°C were not capable of germina­ tion at the optimum temperature after they had been placed on PDA at 40 C for more than 12 hours (see Table 5)« The ascospores of Aycosphaere1 1 a musicola causing leaf spot of banana occurring in the same environment with T. fructigena endured longer a similar treatment (Frossard, 1962). The ascospores germinated after 24 hours incubation at 40°C. The good germination at 20°C (94.0 percent after 12 hours), fairly close to that at the optimum temperature, 2 5 °0 (9 6 .5 percent after 12 hours) (see Table 13) and the high germination of 52.7 percent at 15°0 is another evidence that T. fructigena conidia require lower temperature than most tropical fungi for maximum activity. This behaviour of the conidia might be of some signi­ ficance in nature. The conidia on the fruits, even in the shade, University of Ghana http://ugspace.ug.edu.gh - 153. - could occasionally be subjected to moderately high temperatures over 30°C, that would lower their germination rate considerably. The data of shade Temperature of a cocoa farm at Tafo, Ghana, tabulated by Urquhart illustrate this (see Table 32, below). TABLE 32 Shade Temperature of Cocoa Farm at Tafo, Ghana (l5~year period, 1938-1952.) D a ta ow\ U r y Wqt’V ( iq s s > Month Mean Max. (°F) Mean Minimum (°F) January 91.4 59.1 February 93.7 6 1 .2 March 93.5 6 5 .8 April 93-7 66 .1 May 92.1 66.6 June 89.3 6 6 .1 July 86 .8 66.2 August 8 6 .4 65.O September 88 .0 66.6 October 89 .8 6 6 .3 November 90.5 65 .0 December 90.6 62.7 University of Ghana http://ugspace.ug.edu.gh Several months, it would be observed (June - October) have sufficiently low temperatures favourable to T. fructigena. In the open, the temperature could rise considerably above 30°C and this would be important for T. fructigena formed on coffee berries and banana fruits which usually grow in the open. Lawson _et al. (1970) recorded maximum air temperatures in the open at Kade, Ghana, a moist semi-deciduous forest, of 38°C, 37.6°C and 34.8°C, respectively, at 10cm, 35cm and 150cm above ground level. If the fungus grows in soil as well, bush fires set by hunting gangs are likely to raise the temperature of the ground sufficiently high to destroy the oonidia or prevent germination. Lock and Milburn (1971) have shown that the recorded maximum soil temperature after burning was 59.0°, 3 7 .7° and 3 2 .4°C at the surfaces of the soil and 1 .0cm and 5 .00cm deep, respectively. Light did not have any significant effect. It is even likely that differences observed would be due to normal heterogeneity of fungal spore population. Percentage viability was slightly higher in light (275.0 lux) than in dark after 5, 10 and 20 days' storage at 20°C. Higher percentage survival was also recorded on - 154. - University of Ghana http://ugspace.ug.edu.gh - 155- - some occasions in light (4 6 7 .5 lux) at 28°C (see Table 12). Conidia of T. fructigena were obviously not affected by light intensity of 467«5 lux. It remains to be found whether higher light intensities would have some deleterious effect. Cocoa pods are borne on the trunks and branches of the trees and are always heavily shaded by the closed canopy of the trees. Very little light, therefore, ever reaches them, an intensity o f less than 4 6 7 .5 lux. Some fungi are sensitive to dehydration. Merek and Fergus (1954) found that at 12*- 24°C, the endoconidia of Endoconidiophora fagacearum remained viable longer at 3% than at 7% R.H., and Goos and Tschirach (1962) reported that spores of Gloeosporium musarum survived longest at higher humidities (60 - bCP/o R.H.) than at lower humidities (0 - 2CY/o R.H.). Moreover, the viability of several powdery mildews is best preserved in wet conditions as in the case of conidia of Erysiphe graminis (Metzger, 1942), Eryaiphe polygon! (Yarwood et al., 1954), Oidium heVeae (Corner, 1935) and Phyllaotinia oorylea (jinkora, 1968). T. fructigena conidia belong to this category. University of Ghana http://ugspace.ug.edu.gh - 156. - Humidity was found to be a factor powerfully influencing survival. Conidia of T. fructigena survived longest at 10CY/o R,H. and lost viability very rapidly at any humidity below R.H. It was possible to examine survival over the entire humidity range because no germination occurred even at optimum temperature (25 C). No conidium survived an hour's storage at 0 - QCf/o R.H. In fact, only 1.6 percent of the conidia stored at 8C$> R.H. was viable after only 20 minutes (see Table 7). Reasonable longevity was found at storage humidities above 9Cf?o R.H. Approximately 33 percent conidia survived 24 hours’ storage at 9^fo R.H. and storage for 5 “ 10 days destroyed all the conidia (see Table 10). Survival was considerably better at 100y6 R.H., and survival after 5 days was 93-5 percent and, 66.7 percent after 15 days. Viability was lost rapidly there­ after to give 29.7 percent survival after 20 days and less than one percent survived 40 days’ storage at 10Q$ R.H. (see Table 11). Quick death at lower humidities was accompanied by other events. The conidia apparently possess thin walls that collapsed as water was lost in air (see Plate 5). The rate of shrinkage depended upon the surrounding humidity. Thus all the conidia collapsed in 100 minutes when exposed to an atmosphere of 8CP/o R.H. (see Table 7). The time taken to shrink then lengthened with University of Ghana http://ugspace.ug.edu.gh - 157. - increasing relative humidity (see Figs. 3, 4 and 5). Maximum shrinkage was therefore observed after 9 hours, 24 hours, 15 days and 30 days during storage at 8 5, 90, 95 and 100'/o R.H.., respectively (see Tables 8 - 11). Evidently, humidities at 95S& R.H. and below would encourage loss of water from the spore. As water came out of the vacuoles, the protoplast withdrew from the wall causing a collapse of the wall. It is difficult, however, to imagine how water could be lost by the spore into a humid air of 10Qj&> R.H. and an alternative mechanism might have brought changes in vacuole volume and that of protoplast. The cause of shrinkage of the conidia at 100% R.H. needs further investigation. There are many reports (e.g. Ankora, 1968; Brodie, 1945; Manners and Hossain, 1963; Nour, 1958; Yarwood, 1936) that conidia of powdery mildews collapse v/hen exposed to humidities less than 10C% R.H. It has been suggested that shrinkage may be due both to loss of water by the spore to the air, especially in non-germinated conidia, and its use by the conidium for germination activities. Yarwood (1952) did indeed observe that germinating conidia of Erysiphe polygoni showed greater decrease in volume than shrunken non-germinated conidia. The conidia of powdery mildews do not shrink at 100$ R.H. and conidia of T. fructigena might therefore University of Ghana http://ugspace.ug.edu.gh - 158. - have mechanisms not present in the p#wdery mildew spore. The conidia retained a more stable constitution and survived sW owa ’ey longer in water than at 10C% R.H, as^the contrasting 80.9 percent survival in water after 20 days (see Table 13) and 29.7 percent survival at 10C$ R.H. (see Table 1l). Humid air and liquid water apparently exert completely different influences on the spore. The extremely low germination of the conidia in water, less than 2.0 percent at the optimum temperature (see Table 2) permitted the extension of the survival experiments to examination of the fate of the conidia in water, which has not been possible for spores that survive best at higher humidities as they germinate well in water. The manner in which water is lost from the conidia affected the vigour of the spore. Conidia plasmolysed for 1 hour in 0.7M Potassium nitrate solution (24*6 atmospheres), 0.7M sodium chloride solution (29.9 atmospheres) and 1M sucrose solution (2 5.0 atmospheres) were barely affected showing 89.1, 82.6 and 93*9 percent germination on PM, respectively (see Tables 14 - 16). When a 2.0M sucrose solution was used, germination was still very high, 8 5 .9 percent, on PM (see Table 17)* Conidia from which water had been withdrswn (see Plate 9) would, however, eventually lose vigour. Table 18 shows that an effect was noticeable for immersion, in sucrose solution, exceeding 24 hours. The data may be intepreted as showing that University of Ghana http://ugspace.ug.edu.gh - 159. - death may be due to two causes operating together in air. These are loss of water and a disruption in the functioning of the membranes. The latter does not seem to occur when submerged in fluids and onset of death is delayed, depending on injury caused by loss of water alone. The plasmolysis experiments throw light on the mode of shrinkage of the conidia. Diminishing size of the protoplast as it lost water at humidities below 10($> R.H., or through as yet unknown changes in the protoplasm at 100$ R.H., caused the fragile spore wall to lose its support and to collapse. When the conidium was made to lose water by plasmolysis and- indeed, decreased the volume of the protoplast (see Plates 8 and 9) the wall was held back by the plasmolysing fluid that occupied the space between the plasma membrane and the spore wall. The changes in the protoplast were, however, irreversible. Shrunken conidia floated in water showed distinctly rounded up protoplast and the resumption of the spherical form was due to water that had passed through the spore wall pushing back the infolded wall (see Plates 6 and 7). Shrinkage at humidities below 100$ R.H. is, therefore, a sequel to actual events of death. It, however, affords a most convenient means of assessing viability of the conidia. University of Ghana http://ugspace.ug.edu.gh - 160 . - .&n outstanding feature of most fungal species is the enormous spore production to compensate for the unavoidable waste of reproductive units of all organisms which seems to reach its extreme in the fungi. Vast quantities of T. fructigena are likely to die immediately on production, since the atmospheric humidity of the cocoa farm, even under the canopy never reaches 9Qfc R.H., as again other pertinent data of Urquhart (1955) show in Table 33. It is, however, possible that some of the accumulated conidia on the infected tissues would be protected by overlying conidia and may be found in an atmosphere of considerably higher moisture content. Greater proportion of the conidia might therefore remain viable than would have been expected. The mode of dispersal of the conidia would be critical to their survival. Prolonged exposure would be hazardous. The efficiency of cocoa farm ants, reported to carry the conidia of T. f'ructigena by Dade (1927) is doubtful. The movement of ants is erratic and their coming across a pod is a matter of chance. Rodents which, on the other hand, seek the pods would be more effective dispersal agents. They move directly from pod to pod, ensuring not only quick transfer but also inflicting the wound, which ants cannot do, needed for infection by the fungus. University of Ghana http://ugspace.ug.edu.gh - 1 6 1 . - TABLE 33 Atmospheric Relative Humidity of Cocoa Farm at Tafo, Ghana (13-year period, 1938 - 1952) Data, 'fiom U'rquk<\r,i~ Month Mean % R.H. 9.00 a.m. Mean % R.H. 3.00 p.m. January 85.9 54.1 February 83.7 51.3 March 81.6 54.7 ■alpril 80.5 57.9 May 81.7 6 2 .3 June 84.7 67.9 July 86.0 68.9 August 85-9 6 1 . 3 September 85.1 70.6 October 83.1 69.8 November 81.8 6 5 .4 December 84.2 6 1 .4 University of Ghana http://ugspace.ug.edu.gh Substances in water extract of partially decomposed leaves of soils of farms in which T. fructigena had been found depressed germination (see Table 21). That stimulatory and inhibitory substances exist at the same time in soil, with the latter often predominating, is generally recognised. The failure of the conidia to germinate in the soil extracts (see Chapter M) in this investigation could be due to either very high levels of inhibitory substances or to low nutrient levels. It is possible that both were operating together. Unlike observations of Hora and Baker (1970; 1972), the inhibitory substances here were not volatile as was clearly shown by the experiment in Chapter 0 and actual contact with the spore would be necessary for its operation. These inhibitory substances were fungistatic but not fungicidal. Conidia of T. fructigena which had remained dormant for 1 5 days under the influence of the inhibitory substances of the soil had later been stimulated into germination with nutrient medium (see Table 23). Similar observations had been made by other workers. Lingappa and Lockwood (1961), for example stimulated spores of Fusarium oxysporum f. lycopersici. Clomerella cingulata and Penicillium frequentans which had lain dormant under the influence of the inhibitory substances for 5 days, into germina­ tion with nutrients. University of Ghana http://ugspace.ug.edu.gh The quiescent conidium in the soil faces many hazards. Both organic and inorganic ions in the soil, when present at high levels are likely to impose plasmolytic problems which would shorten longevity. Two other important factors have been demonstra­ ted in this work. The pH of the soil in which the spore is deposited would greatly influence its survival during its exogenously induced dormancy. Survival at the optimum pH of pH 8.0 was more than fifteen fold that at pH 4*0 (see Table 23). Besides, the conidia would face inevitable lytic action of other soil microflora. One of the most ecologically significant result of these experiments was the fact that viable and dead conidia were differently attacked by soil microflora. "While approximately one percent of the originally viable conidia showed signs of attack on the 4th day, as much as 8 5 .9 percent of originally dead conidia had been attacked and 22.1 percent had been completely broken down by lytic aotion (see Tables 24 and 25). The results suggest that the spores produced metabolites that might have affected the microflora around them affording protection. The possibility of substances passing out of spores has also been postulated elsewhere. Lingappa and Lockwood (1962) reported the observation of areas of stimulation of bacteria in the vicinity of fungal spores added to soil. These observations led them to the hypothesis that individual spores might release - 163. - University of Ghana http://ugspace.ug.edu.gh nutrients into the soil, stimulating the growth of antagonistic organisms in their vicinity. The hypothesis proposed here might be valid for a short period as metabolite production could not be expected to be sustained to a rate sufficient to maintain a defensive barrier for more than a few days. The diminishing longe- vity with increasing period of incubation (see Table 24) supports this view. Death of the spore of a fungus would not necessarily lead to immediate attack by soil microorganisms as the result here might suggest. The nature of the wall had been found to be an important factor in spore lysis. Chu and Alexander (1972) found that spores of various fungi killed by ultraviolet light were not equally lysed in soil. Spores of Aspergillus niger and Aspergillus phoenicis were resistant to lysis in soil and only indications of disooloration of the spores 7/ere detected after 2 weeks. By contrast, Colletotricj m lagenarium spores were ruptured to a significant extent by the fifth day. Thielaviopsis basicola spores lost their long side walls as well as cytoplasm within 5 days and Penicillium atrovenetum spores exhibited wall breakage by the seventh day. University of Ghana http://ugspace.ug.edu.gh 4 relation between the melanin content of fungal walls.and their resistance to enzymatic degradation was noted by Potgietcr and Alexander (1966), and quantitative studies in melanin-containing and melanin-free strains of Aspergillus nidulans have confirked this relation (Kuo and Alexander, 1967)- It was found in these studies that synthetic melanin inhibited a gluconase, chitinase and proteinase in vitro and that such a melanin may be very resistant to microbial degradation in soil. The melanin-resistance mechanism is clearly independent of the health of the spore. There are most likely other modes of resistance to lysis. Fungistasis of cocoa farm soil imposed exogenous dormancy on conidia of T. fructigena. Because this fungistasis is capable of modification or elimination by other factors in the environment, e.g. nutrients, it is possible to predict the behaviour of such conidia formed by mycelium in the soil. The survival of such mycelium in soil has been the subjeot of study in this work. The concept of competitive saprophytic ability has been much used recently and many relevant investigations have been carried out. Generally, fungi with very low competitive saprophytic ability do not survive long as saprophytes. Garrett (1950) listed four general attributes that are likely to contribute - 165. - University of Ghana http://ugspace.ug.edu.gh to a high degree of competitive saprophytic ability. They are: (1 ) rapid germination of spores and a high rate of hyphal growth, both favouring rapid colonization; (2 ) good enzyme production, which favours rapid and extensive substrate utilization; (3) production of substances toxic to other organisms, _ which may reduce competition for the available substrates; and (4 ) tolerance of anti-biotic substances produced by other organisms. There is no evidence from the present studies to show that T. fructigena possesses the last two attributes. The greater colonization of the cocoa pod husk baits when the fungus had first access to the bait (see Table 27) indicates that the necessary erzymes were present. The conidia, in addition, germinated quiokly when provided with nutrients (see Table 3 ) and they produced extensive hyphae (see Plate 3). An attempt to increase the nutrient level of the cocoa pod husk baits did not bring uniform rate of colonization. T. fructigena was clearly encouraged well by sodium nitrate only (see Table 28). University of Ghana http://ugspace.ug.edu.gh - 167. - Sodium nitrate benefited T. fructigena because it might be one -of the most suitable nitrogen-compounds for its growth while most probably the other nitrogen compounds favoured other soil micro­ organisms. It remains to be established in further investigations on T. fructigena whether good growth is produced in sodium nitrate- medium. Concentration effects cannot, however, be ruled out. Perhaps at other concentrations both T. fructigena and other soil microflora would behave differently. There are no reasons why T. fructigena cannot colonize other decomposing plant materials, especially the rotting cocoa leaves which litter the farms and form a thick carpet over the ground. Under such circumstances, the survival rate of T. fructigena, although low, in the unamended bait, would be sufficient to ensure continual growth from one rotten leaf to a newly fallen one. Large inoculum without which colonization would be 'unsuccessful could be readily provided by rotted diseased pods with their extensive crop of conidia on the surface and mycelium in the tissues. Studies on survival of a member of the Peronosporales cannot be complete without a consideration of the oospores. An insight of the immense constitutive dormancy of oospores of Peronosporales was University of Ghana http://ugspace.ug.edu.gh provided by Blackwell's (1943a and b) intensive study of the oospores of Phytophthora cactorum. The cospore has an after­ ripening period of 6-8 months at 15° - 20°C. Tabor and Bunting (1923) had shown that T. fructigena oospores are not readily come by. None of the innumerable plates that were cultured during the course of this work produced oospores. Survival of the oospores should be studied when a method to produce the oospores in large quantities has been devised. University of Ghana http://ugspace.ug.edu.gh - 1 6 5 - - VI. SUMMARY 1. Germination of conidia of T. fructigena in distilled water was extremely poor. 2. Germination rates in distilled water at 15° _ 30°C were identical, ranging from 1.0 to 1.4 percent after 48 hours- 3. There was no germination in water at 40°C and only 0.5 percent conidia germinated at 35°C in 48 hours. 4 . On PDA, there was no germination at 40°C in T. fructigena conidia. 5- PDA highly stimulated germination at 15° - 35°C but not at 35° and 40°C. It supported 33-6, 94-0, 96.5 and 30-2 percent germination at 15°, 20°, 25° and 30°C, respectively, after 12 hours. 6. The maximum possible germination was attained in a shorter time at 25°C, the optimum temperature than at 20°C where germination was also very good. 7. The latent period of germination at 25°C was 96 minutes while that at 20°C was 150 minutes. 8. Poor germination of conidia at 35°C, 0.5 percent germination on P M after 36 hours, indicated that conidia were sensitive to higher temperatures. University of Ghana http://ugspace.ug.edu.gh 9. There was no germination after exposure of T. fructigena conidia on PDA to 45°C, even for as short as 3 hours and conidia would not survive incubation at 40°C for more than 12 hours. 10. T. fructigena conidia did not germinate at 80$ - 10($ R.H. 11. Conidia of T. fructigena were extremely sensitive to drying. 12. Conidia of T. fructigena died within less than 20 minutes when exposed to atmospheres of Ofo - ~l&/o R.H. 13. The conidia lived for only 20 and 40 minutes, respectively at 1% and 8C$ R.H. 14. R.H. After 20 days, percentage survival v/as 80.9 and 29.7 percent in water and at 10Q&R.H., respectively. 27. The rate of germination was faster in conidia stored in water than those kept at 100% R.H. for the same time. 28. Plasmolysed conidia showed distinctly rounded protoplast, but did not shrink. 29. 4\ large percentage, above 77 percent, of the conidia remained alive after plasmolysis in 0.7M potassium nitrate, sodium chloride and sucrose solutions for 60 minutes. - 172. - University of Ghana http://ugspace.ug.edu.gh 30. Viability of T. fructigena conidia plasmolysed for 60 minutes by sucrose solutions of higher osmotic pressures of 1.5M and 2.0M was also high; 90.3 and 83.9 percent, respectively. 31. Viability was affected when the conidia were kept in 1.5M and 2.0M sucrose solutions for more than 24 hours. 32. Survival of conidia after 72 hours of storage in 1.5M and 2.0M sucrose solutions was, respectively, 34-1 and 32.7 percent, whilst conidia in distilled water showed 94*7 percent viability. 33* Every conidium observed after storage in sucrose solutions of 1.5M and 2.0M was plasmolysed, though to varying degrees. 34. No germination occurred in non-sterile extracts of soil (pH 9.9) and partially decomposed leaves (pH 8.2) of the cocoa farm. 35* No germination also occurred in autoclaved extracts of soil and partially decomposed leaves. 3 6. Fungistasis could be demonstrated in extract of partially decomposed leaves by its ability to eclipse partially the nutrients of low concentration, N/8 and N/1 6 , of potato dextrose broth. - 1 7 3 .- University of Ghana http://ugspace.ug.edu.gh - 174. - 37. Whilst PDB alone at N/ 1 6 supported 98.6 percent germination, only 1 7 .0 percent of the conidia germinated when the extract was added. The germ tubes were, in addition, very short. 3 8. The fungistatic principle was seemingly non-volatile. 3 9. Approximately 70 percent of the conidia stored in soil extract of pH 8.0 survived 10 days of storage and 51*0 percent was alive after 15 days. That was the highest percentage survival in the pH levels of 4.0, 5*0, 6.0, 7.0, 8.0, 9*0 and pH 10.0. 40. The fungistatic principle was not fungicidal and the conidia survived for several days in the soil extracts. 41. Longevity in the extracts of partially decomposed leaves was closely similar to that of soil, and the conidia were preserved best at pH 8.0 in both extracts. 42. In 'both extracts, pH 4.0 was most unsuitable for storage of T. fructigena conidia. 43* There was considerable difference between the extracts of visible attack by microorganisms of originally viable and dead conidia buried in native soil of 2C$> moisture content. University of Ghana http://ugspace.ug.edu.gh - 175. - 44. On the 4th clay, only 0.9 percent of the 1690 initially viable conidia observed carried a microflora visible under high power of the microscope. 45- Survival after 4 days in the soil was 54.5 percent. 4 6. Initially dead ccnidia showed heavy incidence of saprophytes: (a) Out of 1814 initially dead conidia observed after 4 days of burial in soil, 85.9 percent had been visibly attacked by saprophytes, and; (b) 22.1 percent was completely lysed and could only be identified by their faint outline of their disintegrated walls. 47* Lysis was seemingly prevented, at least for some time, by viable conidia. Wo lysis was detected after 4 days burial in soil. 48. T. fructigena demonstrated limited competitive saprophytic ability. Saprophytic colonization of pieces of cocoa pod husk fell rapidly from 70 percent survival in 10C$ inoculum to 2 percent survival in 50:50 inoculum/unsterilized soil mixture. University of Ghana http://ugspace.ug.edu.gh - 176. - 49. similar trend occurred when pericarp of coffee was used as bait. There was 58 percent survival in 10Q$ inoculum and 4 percent survival in 50:50 inoculum/soil mixture. 50. T. fructigena survived better in cocoa husk at higher percentage inoculum than in coffee pericarp, but this was reversed with increased proportion of unsterilized soil. 51. The position of T. fructigena in relation to its saprophytic competitors w e is clearly indicated to be an important factor. In the 95:5 inoculum/soil mixture 40 percent colonization was recorded for cocoa husk pieces initially coated with inoculum while only 24 percent colonization was recorded for husk pieces initially coated with unsterilized soil. 52. Cocoa pod husk pieces buried without any treatment showed better colonization than the soil coated husk pieces and inferior colonization to inoculum coated husk pieces. 53. Of the three nitrogen compounds viz; ammonium tartrate, peptone and sodium nitrate, sodium nitrate encouraged best survival. University of Ghana http://ugspace.ug.edu.gh - 177. - 54- Approximately 50 percent of the buried cocoa pod husk soaked in O.jfo (50 percent colonization) and 1.5& (56 percent colonization) sodium nitrate solution contained T. fructigena after 4 weeks. 55. Saprophytic survival was slightly less in buried pod husk pieces containing ammonium tartrate, ranging between 20 percent colonization at 2.C% ammonium tartrate to 44 percent coloniza­ tion at 1,0/o a,mrnonium tartrate. 56. T. fructigena did not persist as long in buried cocoa pod husk pieces with peptone. The highest survival was 20 percent, recorded in husk pieces with 0.5% peptone. 57- T. fructigena did not persist extensively in cocoa pod husk pieces with dextrose. Even in 100% inoculum, the best survival after 4 weeks of burial at 1.0% dextrose was 20 percent. 58. £ combination of dextrose (1.0% y / v ) and sodium nitrate (1.5% W/V) highly increased percentage colonization of buried cocoa pod husk pieces by T. fructigena. The fungus persisted in 50 percent of the cocoa pod husk pieces buried in soil containing only 1% inoculum. University of Ghana http://ugspace.ug.edu.gh - 178. - 59. Approximately maximum colonization occurred at 9C$>, 95& and 100^ . inoculum. 60. The implications of the findings have been discussed. University of Ghana http://ugspace.ug.edu.gh - 179. - VII. ACKNOWLED&EftiENT I wish to sincerely express my gratitude to my Supervisor, Dr. G-.C. Clerk, who suggested this problem and guided me with keen interest during the course of this work and for his suggestions during the preparation of this manuscript. My grateful thanks are also due to the laboratory technicians in the Botany Department, particularly to Mr. P.K. Mante and Mr. F.O. Seku, who gave me invaluable technical assistance. I am grateful also to Dr. E.N. Bonaparte of Cocoa Research Institute, Tafo, who provided the large number of cocoa pods used in Chapter R. My gratitude extends to my younger brother and his wife, Mr. and Mrs. Phanuel Maramba of 4 Osney House, Hartslock Drive, London, S.E.2, England, for making possible and financing the printing of all the coloured photographs, without which this thesis would not have been complete, I am also grateful to Air. T. Situ of the Chemistry Department, University of Ghana, Legon, who typed this thesis, and to Mr. Samuel Duodu of the Geography Department, also of this University, for stencilling the graphs. University of Ghana http://ugspace.ug.edu.gh - 180. - Finally, I profoundly thank the Government of Ghana and the Ghana Cocoa, Growing Research Association Limited, Birmingham, England, for financial support without which I could not have pursued this course- University of Ghana http://ugspace.ug.edu.gh - 1 8 1 . - AINSWORTH, G-.C. AND A.S. SUSSMAM (1968). The Fungi. Vol.III. pp. 447 - 486. ACADEMIC PRESS, NEW YORK AND LONDON. ANKORa, J.K. (1968). Studies on the release, survival and germination of conidia of Phyll^ctinia ccrylea (Pers.) Karst. M.Sc. THESIS, UNIVERSITY OF GHANA. ARAGAKI, M., R.D. MOBLEY AND R.B. HINE (1967). Sporangial germination of Fn.ytophthora from Papaya. MYCOLOGIA, 59: 93 - 102. BAILEY, N.T.J. ("1959) • Statistical Methods in Biology, pp. 78 - 99. THE ENG. UNIVERSITY PRESS, LONDON. BLACKWELL, ELIZABETH (1943a). The life history of Phytophthora cactorum (Let. & Cohn) Schroet. TRANS. BE. MYCOL. SOC., 46: 385 - 392. --------, (1943b). On germinating the oospores of Phytophthora cactorum. TRiNS. BR. MYCOL. SOC. 26: 71 - 89. BOATEFG, E.A. (1960). A Geography of Ghana, pp.27 - 32. CAMBRIDGE AT THE UNIV. PRESS. BOURIQUET, &. (1959). a) Annual Report of the Cameroons Develop­ ment Corporation; ¥) Plant diseases and pests in some African territories. REV. APP. MYCOL., J8: 656. BRIAN, P.W. (9160). Antagonistic and competitive mechanisms limiting survival and activity of fungi in soil. IN THE ECOLOGY OF SOIL FUNGI, PARKINSON D. AND J.S. WAID ed. pp.115 - 129, LIVERPOOL UNIVERSITY PRESS. BRODIE, H.J. (1945)« Further observations on the mechanism of germination of the conidia of various species of powdery mildew at low humidity. CANAD. J. RES. C., 2J5: 198 - 211. BRUN, J. (1954)* Rotting of Bananas in the French Cameroons. FRUITS D'OUTRE MER, 2 : 311-313. , (1955)- Phytopathology. REV. APP. MYCOL., 34: 433. V I I I . LITERATURE CITEL University of Ghana http://ugspace.ug.edu.gh - 182 . - BRUN., J. OND G-. MERNY (1947). Sur une pourriture nouvelle des bananes Gros Michel. FRUITS D'OUTRE HER., 2: 37 “ 42. BRUN, J. AND J. CHAMPION (1955). Banana cultivation in the Br. Cameroons. FRUITS D'OUTRE MER., 10: 163 - 170. BUNTING, R.H. (1923; 1924). Ideological Notes. JOURN. GOLD COAST AGRIC & COM. SOC. ii (3-4): PP. 1 6 3 - 165 (1923). Cited in REV. APP. MYCOL. , III: p.325 (1924). BUTLER, F.C. (1953). Saprophytic behaviour of some cereal root-rot fungi. III. Saprophytic survival in wheat straw buried in soil. ANN. APPL. BIOL., ij-O: 305 “ 311* ----- , (1959). Saprophytic behaviour of some cereal root-rot fungi. IV. Saprophytic survival in soils of high and low fertility. ANN. APPL, BIOL., 28 - 3 6. BUXTON, JEAN WARD AND A.G.P. BROWN (1962).. Roth&msted Experimental Station Report. Cited in REV. APP. MYCOL., 42: (1963) p.653. CHAMBERS, S*C. AND N.T. FLENTJ^ j (19 6 9). Relative effects of soil nitrogen and soil organisms on survival of Ophiobolus graminis. AUST. J. BIOL. SCI., 22: 275 - 278. CHINN, S.H.F. (1953). A slide technique for the study of fungi and actinomycetes in soil with special reference to Helminthosporium sativum. CAN. J. BOT., 31,: 718 - 724. CHU, B. STELLA AND M. ALEXANDER (1972). Resistance and suscepti­ bility of fungal spores to lysis. TRANS. BR. MYCOL. SOC., 38: 489 - 497. CLERK, G.C.. (1972). Germination of sporangia of Phytophthora palmivora (Butl.) Butl. ANN. BOT,, 3 6 : 801- 607. CLERK, G.C. iND M.F., MADELIN (19 6 5). The longevity of conidia of three insect-parasitizing Hyphomycetes. TRANS. BRIT. MYCOL. SOC., 48: 193 - 209. University of Ghana http://ugspace.ug.edu.gh - 183. - COCHRANE, V.W. (1958). Physiology of Fungi. 524pp. JOHN WILEY 4ND SONS, INC., NEW YORK. ------- , (1966). Respiration and spore Germination. IN THE FUNGUS SPORE, COLSTON PAPERS No.18, M.F. MADELIH ed., pp.201 - 215. LONDON: BUTTERWORTH. CORNER, E.J.H. (1935)- Observations on resistance to powdery mildews. NEW PHYTOL., 34: 180 - 200. DADE, H.A. (1927). Economic significance of cacao pod diseases and factors determining their incidence and control. REV. APP. MYCOL., VI: 657 & 659. , (1932). Further observations on Cacao pod diseases in the Gold Coast. REV. AFP. MYCOL., 11_: 701. de BRUYN, H.L.G. (1926). The overwintering of Phytophthora infestans (Mont.) DeBy. PHYTOPATHOLOGY, 1_6: 121 - 140. DILLON-WEST ON, W.A.R. AND E.T. HALNAN (1931). The reactions of certain wave lengths in the visible and invisible spectrum. II, Reactions of urediniispores to visible light: Wave lengths between 400 and 700H /i. PHYTOPATH. ZEITS., 4: 229 - 246. ^ ----------- , (1932). Effect of ultraviolet radiation on the urediniospores of black stem rust, Puccinia graminis tritici. SCI. A.GR., 12: 81 - 8 7. DOBBS, C.G. AND M.J. GASH (19 6 5). Microbial and residual mycostasis in soils. NATURE, LONDON, 207: 1354 - 1356. DOBBS, C.G. AND V/.G. HINSON (i953) - A widespread fungistasis in soils. NATURE, LONDON, 172: 197. FERGUS, C.L. AND R.D. SCHEIN (i960). Effect of environment on germination of ascospores of Urnula craterium MYCOLOGIA, 52: 719 - 725. University of Ghana http://ugspace.ug.edu.gh FROSSARD, P. (1962). Influence de la temperature sur la projection et la viabilite' des as cospores de Mycosphaerella musicola Leach'. FRUITS, 17: 382 - 38 5. GARRETT, S.D. (1938). Soil conditions and the take-all disease of wheat. III. Decomposition of the resting mycelium of Ophiobolus graminis in infected wheat stubble buried in the soil. ANN. APPL. BIOL., 2j5: 742 - 766. ------, (1950). Ecology of the root-inhibiting fungi. BIOL. REV., 2£: 220 - 254. ------, (1970)* Pathogenic root-infecting fungi. CAMBRIDGE UNIV. PRESS. 294PP. GOOS, R.D. M. TSCHIRSCH (19 6 2). Effect of environmental factors on spore germination, spore survival and growth of Gloeosporium musarum. MYCOLOGIA, LIV: 353 “ 3^ 7* GOTTLIEB, D. (1950). The Physiology of spore germination in Fungi. BOT. REV., 16: 229 - 257. -------, (19 6 6). Biosynthetic processes in germinating spores. IN THE FUNGUS SPORE, COLSTON PAPERS No.18, M.F. MADELIN ed., pp.217 - 234. LONDON: BUTTERWORTH. GREGORY P.H. (1969). Blackpod disease project Report, pp. 1 - 52. NEILL. HARRISON, C.H. (1942). Longevity of the spores of some wood- destroying hymenomycetes. PHYTOPATHOLOGY, 32: 1096 - 1097. HART, H. (1926). Factors affecting the development of flax rust, Me lamps ora lini (Pers.) Lev. PHYTOPiiTH., 1_6: 1 8 5 - 205. HORA, T.S. AND R. BAKER (1970). Volatile factor in soil fungistasis. NATURE, 22£: 1071 - 1072. University of Ghana http://ugspace.ug.edu.gh - 1 8*. - HORA, T.S. AND R. BAKER (197^ )« Soil fungistasis: microflora producing a volatile inhibitor. TR/iNS. BR. MYCOL. SOC., j>9: 491 - 500. HYRE, R.A. AND R.S. COX (1953). Studies in the Physiology of Phytophthora phaseoli (Abs.) PHYTOPATH., ^3: 41 9 - 425* INGOLD, C.T. (1953). Dispersal in Fungi. CLARENDON PRESS. OXFORD. 208 pp. JACKSON, R.M. (1958a). An investigation of fungistasis in Nigerian soils. J. GEN. MICROBIOL., 18: 248 - 258. — , (1958b). Some aspects of s*il fungistasis. J. GEN, MICROBIOL., 1_9: 390 - 401 . KUO, M.J. AND M. ALEXANDER (1967) - Inhibition of the lysis of fungi by melanins. J, BACTERIOL., 9^.: 624 - 629. LAWSON, G.W., K.O. ARMSTRONG-MENSAH AND J.B. HALL (1970). A catena in Tropical Moist Semi-Deciduoxse Forest near Kade, Ghana. J. ECOL., j>8: 371 - 398. LEACH, (1953; 1954). Unpublished reports. Cited by WARDLAW, C.W. (1972). Banana Diseases including Plantains and Abaca. 2nd ed., 878pp. LONGMANS LONDON. LINGAPPA, B.T. AND J.L. LOCKWOOD (1961). The nature of the wide­ spread soil fungistasis. J. GEN. MICROBIOL., 26: 473 - 4 8 5. ” J ' “ (1962). Relationship of soil microbes to the widespread soil fungistasis (Abs.) PHYTOPATHOLOGY, 52: 739. LOCK, J.M. 4ND T,R. MILBURN (1971) • The seed biol*gy of Theme da triandra Forsk. in relation to fire. 11TH SYMPOSIUM OF THE BRITISH ECOLOGICAL SOCIETY, UNIVERSITY OF EAST ANGLIA, NORWICH, 7 - 9 JULY, 1970. 337 - 349. University of Ghana http://ugspace.ug.edu.gh - 186. - MACER, R.C.F. (1961). The survival of Cercosporella herpotrichoides Fron in wheat straw. ANN. APPL. BIOL., 1+9'- 1 6 5 ~ 172. MALLAMAIRE, A. (1934). The year's phytopathology on the Ivory Coast. AGRON. COLON. XXIII (202): 114 -119. MANEVAL, W.E. (1924). The viability of uredospores. PHYTOPATH., lij.: 403 - 407. MANNERS, J.G. AND S.M.M. HOSSAIN (1963)- Effect of temperature and humidity on conidial germination in Erysiphe graminis. TRANS. BRIT. MYCOL. SOC., 4 6: 225 - 234- M.1RK2TT, C-L. (1953). Lethal and mutagenic effects of ultraviolet' radiation on G-lomcrolla oonidia. EXPTL. CELL RES., 427 - 435. MEREDITH, D.S. (i960). Some observations on Trachysphaera fructigena Tabor & Bunting, with particular reference to Jamaican bananas. TRANS. BRIT. MYCOL. SOC., l£ : 100 - 104. MEREK, E.L. AND C.L. FERGUS (1954). The effect of temperature and relative humidity on the longevity of spores of the oali wilt fungus. PHYTOPATH., 4^: 61 - 64. METZGER, I. (1942). Versuche zur Aufbewahrung lebender Sporen von Weizen-und Gerstenmehlau. KUHN-ARCH., 6^: 163 - 172. McCREA, A. (1923). Longevity of spores of Aspergillus oryzae and Rhizopus nigricans. SCIENCE, 58: 426. McKAY, R. (1935). Germination of resting spores of onion mildew (Peronospora schleideni). NATURE, 135: 306 - 307. MORSTATT, H. (1936; 1937). Coffee pests and diseases in Africa (Continuation). TROPENPFLANZER XXXIV (11) : (1936), pp.455 - 481. Cited in REV. APP.' MYCOL., _l6: (1937) p.247. NAQVI, S.H.Z. /iND H.M. GOOD (1957)• Studies of the Aging of conidia of Monilinia fructicola (Wint.) Honey. CAN JOUR. BOT., 635 - 645. University of Ghana http://ugspace.ug.edu.gh - 187. - NOUS, (1958). Studies on Leveillula taurica (Lev,) Arn. and other powdery mildews. TRANS. BRIT. MYCOL, SOC., 41.: 17 - 37. POTSfKEBR, H.J., AKD H. ALEXANDER (1966). Susceptibility and - resistance of several fungi to microbial lysis. J. MCTERIOL., 91.: 1526 - 1532. RESPLANDY,.Mme. R., J. CHEVAUGEON, M. DELASSUS AND M. LUC (1954). First annotated list of parasitic fungi of cultivated plants in the Ivory Coast. AM. INST. RECH. AGRON., SER. C. (ANN. EPIPHYT), jj(l): 1 - 61. ROGER, L. AND A. MALLA&AIRE (1937) - Notes on African phytopathology. ANN. AGRIC. AFR. OCC. i(2): 187 - 206. ROSEN, H.R. AND L.M. WEETMAN (1940). Longevity of urediospores of crown rust of oats. ARKANSAS UNIT. (Fayetteville), AGRIC. EXPT. STA., BULL., 3 ~ 20. Cited in AINSWORTH, G.C. AND A.S. SUSSMAN (1968J7 The Fungi. Vol.III, p p . W - 486. ACADEMIC PRESS, NEW YORK AND LONDON. SIMMONDS, N.W. (1960). Bananas. REV. APP. MYCOL., 431 - 434 & 727. SOLOMON, M.E. (1952). Control of Humidity with Potassium hydroxide, sulphuric acid or other solutions. BULL. ENT. RES., 42: 543 - 554- STOVER, R.H. (1958). Studies on Fusarium wilt of bananas. III. Influence of soil fungitoxins on behaviour of Fusarium oxysporum f. Cubense in soil extracts and diffusates. CAN. J. B0T.,“3^T 439 - 453. SUSSMAN, A.S. (1966). Types of dormancy as represented by conidia and ascospores of Neurospon,. IN THE FUNGUS SPORE, COLSTON PAPERS No.18, M.F. MADELIN ed., pp.235 - 257. LONDON BUTTERWORTH. TABOR, R.J. AND R.H. BUNTING (1923). On a disease of Cocoa and Coffee fruits caused by a gungus hitherto undescribed. ANN. BOT., LONDON, 3 7 : 153 - 1 5 7 . University of Ghana http://ugspace.ug.edu.gh - 188. - TEITELL, TE YE GAGA , THOROLD, TINLINE, TURNER, P URQUHART, WOLF, F.A WOLF, F.A YARWOOD, L. (1958). Effects of relative humidity on viability of conidia of Aspergilli. AMER. J. BOT., 4£: 748 - 753* A. G.C. CLERK (1972). Germination and survival of conidia of Cercospora canescens Ellis et Martin. TROP. AGRIC. (TRINIDAD) Vol.49, No.3, pp. 197 - 204. C.A. (1956). Report on the Plant Pathologist. ANN. REP. DEV. AGRIC. (Central) NIGERIA, 1953“54, Part II: 36-41. R.D., J.F. STAUFFER AND J.G. DICKSON (i960). Cochliobolus sativus. III. Effect of ultraviolet radiation. CAN. J. BOTANY, 38: 275 - 282. .D. AND N.K. LOVI (19 6 2). Outbreaks and new records (W. Afr. Cocoa Res. Inst., Tafo, Ghana). REV. APP. MYCOL., 41: 358. D.H. (1955). Cocoa, pp.230. LONGMANS, GREElj;, LONDON. (1961). Cocoa 2nd ed., pp.293- LONGMANS, LONDON. . AND WOLF, F.T. (1947). Effect of temperature on germination. THE FUNGI II: 221 - 235. JOHN WILEY & SONS INC., NEW YORK. . (1934). Downy mildew of tobacco. PHYTOPATH., 24.: 337 - 363. C.E. (1936). The tolerance of Er.ysiphe polygoni and certain other powdery mildews to Low Humidity. PHYTOPATH., 2 6 : 845 - 859. (1952). Some water relations of Er.ysiphe polygoni conidia. MYCOLOGIA, 44: 506 - 522. YARWOOD, C.E., S. SIDKY, M. COHEM AND V. SANTILLI (1954). Temperature relations of powdery mildews. HILGARDIA, 22: 603 - 622. University of Ghana http://ugspace.ug.edu.gh - 189. - IX. ANONYMOUS PUBLICATIONS (in chronological order) 1. (1921) "Report on the Agricultural Department, Government of the Gold Coast, for the year 1921, p.77". REV. APP. MYCOL., II: p.203, (1923). 2. (1935) "Regulations made under the Importation of Plants Regulation Ordinance (applicable to the Colony and Protectorate of Nigeria, including the Cameroons under British mandate) 1935. No.4 of 1936 - 6pp.". REV. APP. MYCOL., VS: p.79, (1937). 3. (1936) "Importation of Plants Regulation Ordinance, Gold Coast, No. 18 of 1936 Regulations No.25 of 1936 - 6pp.". REV. APP. MYCOL., 16: p.351, (1937). 4. (1952) "Map No.249 of the Distribution Maps of Plant Diseases, 2nd edn. 1. zi (1957), by Commonwealth liycological Institute". REV. APP. MYCOL., 22: p.288 (l953). 5. (1956 - 1958) Ann. Reps. Cameroons Development Corporation 1954, 1956 and 1957. REVS. APPL. MYCOL., ^ p.278 (1956); 36: p.638 (1957) and 22.: p . 636 (1958), respectively. University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E C O L L A P S E _ P E R C E N T A G E G E R M IN A T IO N TIME OF S T O R A G E IN OAYS FIG. 5 EFFECT OF RELATIVE HUMIDITY ON LONGEVITY OF CONIDIA OF T. FRUCTIGENA STORED IN LIGHT AT 25° C a ■ 75'/. R.H □----□ 0 0 % R.H *----A837. R.H University of Ghana http://ugspace.ug.edu.gh P E R C E N T A G E G E R M IN A T IO N - 6 7 . - FIG. 6 CORRELAT ION BETWEEN PERCENTAGE COLLAPSE AND PERCENTAGE SURVIVAL OF CON ID IA OF T. F R UC T IG ENA STORED AT VARIOUS RE LAT I VE HUMIDIT IES AT 2 5 ° C ( DATA FROM T A B L E 8 TO II ) • , 8 3 % R.H. 0 , 9 0 % R.H. A , 9 5 % R . H o , 1 0 0 % R.H. University of Ghana http://ugspace.ug.edu.gh PE RC EN TA G E SU RV IV AL OF T. FR U C TI G E N A lOOp 8 0 - 60 - 4 0 - 20 - 0 _ 70 FIG. 12 - 1 3 3 . - PERCENTAGE INOCULUM COLON ISA T ION OF B A I T (CACAO POD HUSK ) BY T. F R U C T IG E N A P LACED A T VAR IOUS P O S I T I O N S FROM T H E B A I T S • ----------• BAITS COATED WITH INOCULUM A --------- A B A I T S COATED WITH SOIL o --------- o NON -COATED BAITS University of Ghana http://ugspace.ug.edu.gh