University of Ghana http://ugspace.ug.edu.gh This is to certify that the work presented in this thesis: "Studies on the physiology of the different sized sporangia of Phytophthora l!!!!!!!2!! (Butl.) Butl." was done entirely by me in the Botany Department. University of Ghana, Legan, from September 1975 to September 1977. This work has never been presented either 1n part or completely. for any degree of this University or elsewhere. ~ . Comfort Mavis ~0c. (Hons) University of ch;t; Legon. ~~ PROJiSSOR G.e.CLERK SUPERVISOR University of Ghana http://ugspace.ug.edu.gh STUDIES ON THE PHYS IOLOGY OF mE DIFFERENT SIZED SPORAt«;JA OF PHYTOPBTHORA PALMIVORA (Btrl'L.) Bt1l'L. A Theail presented by COMPORT !t\VIS ASOMl\NING B. Sc. (BoDS) in partial fulfilment of the requirements for the M.Sc. Degree of the University of Ghana September. 1977. Prom: 'l'he Department of Botany University of Ghana Legon. University of Ghana http://ugspace.ug.edu.gh ABSTRACT sporangia of Phytophthora pnlmivora varied greatly in size. Sporangial lengths which ranged from 12.5 to 60.5 }Am were used to separate the sporangia into three size-class, viz., Small - less than 24.5 \.1m, Medium - 24~5 to 48.5 um and Large - lIIOre than 48.511m in length. Variation in widths of the sporangia was not extensive (15 - 30 pm) and was not considered a useful criterion for defining size-classes. , All the media used, except C8ssava-dextrose agar, encouraged the formation of Medium·sized sporangia in greater quantities, ranging from 52.5 per cent on Glucose-Asparagine medium to 93.3 per cent on Glucose-Peptone medium, than Small-sized and Large-sized sporangia. Approximately 61 per cent of the sporangia formed on Cassava-dextrose agar were less than 24.5 pm in length. Other media on which Small-sized sporangia were formed in considerable quanti- ties, ranging from 16 to 30 per cent, were HBlt-dextrose agar, potato-dextrose agar, yam-dextrose agar and Glucose-Ammonium tar- trate. 'lbe sporangia formed on pods of Amelonado, T85/799, T63/967 and T79/467 cocoa clones and on Glucose-Asparagine medium consisted of considerable proportions, 20.0 to 47.1 per cent, of Large-sized sporangia. Vegetative growtq, degree of a.poraiat'f.tfr1 2Qd sporangium formation, were also influenced by the type of carbohydrate and nitrogenous compound supplied. P. palmivora formed far greater number of sporangia on medium with Glucose as carbon-source than on media containing Fructose, Galactose and . Sucrose. Ammonium tartrate, Asparagine and Peptone were also inferior to Sodium nitrate as Nitrogen sources. Sporangia of different sizes, supported by endogenous substances alone, showed varying germination capacities. At 220 C, the Medium-sized and Large-sized sporangia germinated better by means of zoospores, 89.7 and 96.0 per cent, respectively, than the Small-sized sporangia, 72.1 per cent. 'lbe number of zoospores in a Small-sized sporangium averaged nine; there were 15 zoospores in a Medium-sized sporagium and 19 in a Large-sized sporangium, which were larger in size than those of the other two Size-classes. The ge~m tubes of the Medium-sized and Large-sized sporangia formed at 30 C in distilled water grew to a maximum length of 1408 _ 1449 \lm whilst those of the Small-sized sporangia attained a maximum length of 1278 \.Im~ The number of germ tubes emerging from a sporangium increase With increase in sporangial size, University of Ghana http://ugspace.ug.edu.gh Sporangia of different sizes responded differently to nutrients. The Large-sized and Mediam-eized sporangia were stimulated during germination to a grenter extent by casein hydrolysate, Peptone, yeast- extract, Ammonium tartrate (only at 300 C during direct germination), cocoa pod extract and cocoa root exudate. Analysis showed that cocoa root exudate of a 20.oay old seedline contained the carbohydrates Galactose, Glucosamine, Glucose, Lactose and Ribose and the amino acids, Alanine, Ammonia. Asparagine, Glycine, Histidine, Isoleucine, Leucine, Serine, Threonine, Tyrosine and Valine. The number of germ tubes formed in the nutrient media was not related to sporangial size. A greater percentage of small-sized sporangia produced a larger number of germ tubes per sporangia (four or more) in 1.0 per cent Caaein hydrolysate solution than Large-sized sporangia. The reverse occurred in solutions of 0,5 per cent Casein hydrolysate and 1.0 per cent Yeast- extract. Small-sized sporangia were severely depressed during indirect germination by CeCl2 and (~)2BPOt. solutions, both at 2.0 - 5.0 mH. Desiccation caused by exposure to 951. R.H. for one minute, on the other hand . destroyed the Medium and Large-sized sporangia more quickly than the Small-Sized sporangia. All the sporangia were, however, killed by five minutes exposure to 951. R.H. Sporangia of P. palmivora of all sizes contained the same densities of lipids. proteins, mitochondria and R.N.A. University of Ghana http://ugspace.ug.edu.gh 11 CONTENl'S Page 1. INTROJ)OOTIDN 1 11. LITERATURE REVIEW 4 :UI. *TERJAI8 AND GENiML J9mlODS 8 i. MIl teria1. 8 11. General Methode 8 a. Maintenance of Stock Culture 8 b. Production of Sporangia on Cocoa pods 8 c. Methods of Sterilization 9 d. Culture of !. palmlvora on nutrient media 10 eo Sporangial Germination Methods 10 f. Aue8llment of Growth of Culture 11 ,. Aaseaament of Sporulation 11 h. Assessment of Sporangia1 Germination 11 i. Preparation of Cocoa Pod Extract 12 jo Chemicals 13 k. Culture Media 13 1. Measurement of pH of Media 14 Ill. Staining Methods 14 1. Ribonucleic acid (RoN.A.) 14 2. M:L tochondria IS 3. Lipid. IS Proteina 16 University of Ghana http://ugspace.ug.edu.gh iii page n. Analysis of Sugars by paper CbJ;omatography 17 1. Application of test solution and subsequent development of the chroma togram 17 2. Detection of sugars 18 3. Identification of developed spots and estimation of concentration of respective substances 18 o. Humidity Chambers 19 p. Maintenance of Constant Relative Humidities 19 q. Statistical Analysis 19 r. Experimental Precautions 21 IV. RESULTS A. RelatiolUlhip between the lengths snd widths of Sporangia of !. palmivora 22 B. Categorization of the Sporangia into size- classes 25 C. Size of Sporangia of !. palmivora formed on different nutrient media 32 D. Influence of carbohydrates and nitrogenous compounds on Sporangia1 s be 42 I. Size of Sporangia of !. palmivora formed on pods of different cocoa clones 58 F. Position of Sporangia of different sizes on sporulating hyphal branch 65 G. Indirect Germination of Sporangia of different sizes 71 University of Ghana http://ugspace.ug.edu.gh iv Page H. Productive capacity of Sporangia of different sizes 74 I. Direct Germination of Sporangia of different sizes 79 J. Amount of hyphal growth supported by endogenous substrates of Sporangia of different sizes 83 ~ Formation of secondary Sporangia by Sporangia of different sizes 88 L. Germination of Sporangia of different sizes in Cocoa pod extract 93 M. Effect of Cocoa root exudate on germination of Sporangia of different 8izes 96 N. Analysis of root exudate of cocoa 101 o. Effect of nitrogenous compounds on germination of Sporangia of different sizes 107 P. Development of Germ tubes by Sporangia of different sizes in nutrient media 113 Q. Germination of Sporangia of different sizes in CaC12 and (NHt.>2BP04 solutions 116 R. Mitochondria. R.N.A. snd Nutrient Reserves in Sporangia of different sizes 120 S. The Susceptibility of Sporangia of different sizes to desiccation 125 V. GENERAL DISCUSSIOlt! ••• _ 130 VI. SUMMIUty lSI VII. ACIWLEDGEHEN'l'S 159 VIII. LITERATURE CITED 160 University of Ghana http://ugspace.ug.edu.gh I. I N T ROD U C T ION The genus Phytophthora de Bary is made up of very important parasites which together affect an extremely wide range of host species including trees, shrubs and herbaceous plants from many families of flowering plants and conifers. Some Phytophthoras are confined to a limited number of host species. Thus, Phyt~phthora infestans (MOnt.) de Bary attacks only members of the Solanaceae. In contrast, a number of Phytophthoras infect a great range of hosts. Phytophthora palmivora (Butl.) But1. and Phytophthora cinnamoni Rands are prominent examples of this group~ Chee (1969) listed 138 host plants attacked by P. palmivora and Zentmyer and Thorn (1967) provided a larger host list of 207 for P. cinnamoni. It is strongly believed that there are many more hoats of P. palmivora not yet observed among the wild flora. In Ghana, agriculturalists and plant pathologists are very much preoccupied with the damage caused by P. palmivora to cocoa (Theobroma ~~Llnn.) and the poten~ial of the fungus as a serious pathogen of other valuable crops is often overlooked. Indeed, P. palmivora has not been sought on these hosts although many local economic crops are reported host plants elsewhere. P, palmivora, for example, causes seedling blight or die-black of ~ ~ Osbeck (Ocfem1a and :Roldan; 1927), avocado (fe~ americana Mill.) (Cono- ver, 1948) and cashewnut (Anacardium occidentale Linn.)(Kumararaj snd Bhide, 1962); foot-rot of black pe?per (Piper nigrum Linn.) University of Ghana http://ugspace.ug.edu.gh 2 (Thompson, 1940)and pineapple (~ sativus Schult.) (Mehrlich, 1934). and fruit-rot of papaya, pineapple and tomato (Lycopersicum esculentum Mill.) (Chee, 1969). ~lmivora habouring on these hosts could form a source of inoculum for cocoa infection. Asexual reproduction by sporangia undoubtedly constitutes the main route of propagation of the parasite and the sporangium there- fore remains the principal agent of infection. Some aspects of the physiology of the sporangia, however, still remain unexamined and their importance in epidemiology is not known. One such aspect 1s the respective contribution of sporangia of different sizes during infection, since P. palmivora in common with Some PhytDph~8S produ- ces sporangia of very different sizes. Despite observations of the influence of medium, host, age of culture, moisture and light on size and shape of sporangia of P. palm1vora (Waterhouse, 1974) the literature contains no information on whether the different morpholo- gical forms are different physiologically. The present studies were carried out to ?rovide some informa- tion on the physiology of ~lmivora sporangia of different sizes anc to extend knowledge on the effect of medium and host on the morphology of the sporangia. This investigation is important because there is strong likeli- hood that some of the dramatic variation in sporangial morphology by hosts of other Phytophthora species may exist in P. palmivora which has such uncommonly large host range. If differences in physiology occur amnne> R""""\~gia of different sizes, the relative importance University of Ghana http://ugspace.ug.edu.gh 3 of host spEcies and varieties of hosts on which the most vigorous sporangial types are produced would be identified. Leonian (1927) provided one of the most illustrative examples of host effect on sporangial morphology. He inoculated fruits of banana (~ sapientum Linn.) egg plant (Solanum melongena Linn.) lemon (Citrus !!!E!_~ L., var. !ti~), orange (£.~r.2!. aurant1um Linn.), pepper (capsi~ Linn.) and tomato and tuber of potato (~ tuberosum Linn.) with ~tophthora capsici Leonian, Phytophthora phaseoli Thaxter, Phytophthora omnivora de Bary and Phytophthora mexicana Hotson snd Hartge. In spite of the ready infection of all hosts, sporangia were produced in great abundance only on tomatoes, egg-plants and pe?pers. Whenever the sporangia occurred, however, there were striking differences in their morphological features, their Size, shape and nature of their papillae. The fungi commonly formed small-sized sporangia on tomato and large-sized s?Qrangia on egg-plant. Shapes of the sporangia included numerous forms, typical lemon-shape, globular, ob?yriform, ellipsoidal and filiform shapes. On tomato, the sporangia of ~.~e~, for instance . were small and globular whilst those on egg-plant and pepper were ellipsoidal and large. The germi- nation capacity and physiology of the various forms were not investi- gated. University of Ghana http://ugspace.ug.edu.gh 4 II. LITERATURE REVIEW The references in the 1itaratu~e on physiology of ~vo~ concerned studies that examined the s?orangial population as a whole from cultures or host plants. There is, therefore, apparently no information whatsoever on the behaviour of sporangia of different sizes. Turner (1960) found that soil extracts permitted copious zoospore formation in P. palmivora. He later observed that CTurner. 1963) 68 and 78 per cent of P. palmivora sporangia produced zoospores in water and root exudate of cocoa, respectively. The ability of the sporangia to form zoospores in root exudates of twenty other flower- ing plants was also demonstrated. Tarjot (1967) obtained maxLmum zoos Clore discharge in water at an optimum temperature of 15 - lSoe within 60 - 90 minutes. Bimpong (1969) found during her work on zoospore motility and chemotaxis in P. pslm~ that sporangia of the same age formed in air gave differential germination rates. When the sporangia were incubated in water at 22°e to obtain zoospores, she observed that sporangia from the same inoculated cocos pod and of the same age (about 24 hours) might give as low as zero per cent germination or as high as 95 - 100 per cent. Halm (1970) confirmed the erratic germina- tion behaviour of the sporangia 1n tests with distilled water and nutrient (amino aCid, carbohydrates and proteins) solutions. University of Ghana http://ugspace.ug.edu.gh 5 Extensive investigations by Clerk (1972) showed that p~ ~lmi­ !2!:! sporangia formed zoospores in distilled water at 10 - 34°C with an optimum at 22°C and germinated by means of germ tubes at the higher temperatures, 30 - 34°c. Zoospore formation in distilled water at 220 C was extremely high, 96.6 per cent. Zoospore formation at this temperature was depressed to varying degrees by nutrients, including cocoa pod extract, and mineral salts. Direct germination was on the other hand encouraged by the nutrients at 22°C. The primary germ tubes of sporangia germinating in rich nutrient solu- tions usually developed into prominent swellings immediately on emergence from which numerous secondary germ tubes arose. Clerk U974) later showed during ultrastrcctural studies that the swellings contained typical hyphal organelles. The sporangia of ~~lm~ always develop flagella (Hel!lDes and Hohl, 1969; Clerk, 1974). During direct germination, the flagella which then have no apparent function break down. Hemmes and Hohl (1972) described the processes involved. There was first the cleavage of the ensheathing flagellar vacuoles into small vesi- cles, thus leaving the axonemes unenclosed in the cytoplasm, In a second stage the axonemal microtubules lost their parallel arrange- ment and gradually disintegrated with the central microtubules breaking down first. Some of the directly germinating sporangia of P. palmivora produce secondary sporangia at the apices of short germ tubes. University of Ghana http://ugspace.ug.edu.gh 6 Two majo~ nuclear events/ mitosis and nuclear degeneration, which occurred simultaneously within the spor~um, have been described by Hemmes and Hohl (1973). Mitotic division was i~tranuclear with the nuclear envelope intact throughout the division process. Division involved centrioles and e~centrically located spindle. There was a persistent localized dense zone on the inside of the nuclear envelope containing short intranuclear microtubules near the centrioles during all stages of the nuclear cycle. While these events were taking place, other nuclei within the same sporangium become surrounded by membranous material and degenerated by an autophagou8 process. Hemmes and Hohl suggested that the disintegration of nuclei provides speci- fic precursors for mitosis or that the removal of nuclei helps to maintain a constant nuclear-cytoplasm ratio by counter-balancing the numerical increase of nuclei as a result of the mitosis. Wang and Bartricki-Garcia (1973 - 1974) recently studied the nature of reserve carbohydrates of P. palmivora sporangia. The cyto- plasm is rich in soluble P- (1-+ 3) -glucans of which two types could be recognized, neutral glucan and the major component, phosphogluCdna; which together constitute 20 - 25 per cent dry Weight of the sporan- gium. The phosphoglucans are present in the sporangium and zoospores but not in the myceli~ and the sequence of apl~arance and disappea- rance of these compounds c1uring the asexual life cycle of EL~lmi­ ~ suggests a major role of these polymers in biochemical diffe- rentiation. University of Ghana http://ugspace.ug.edu.gh Tarjot (1974) obtained in his measurements, lengthS of sporangia of f~~ ranging from 35 to 50 )JIll and widths from 23 to 28 ~Im. He, however, detected that sporangia taken from the bottom of a cul- ture slant in a test tube were much larger (average length 52 ~m. average width 34 ~m) than those taken from the upper part of the same tube (average length 45 ~m, average width 29 ~m). He also noted that the production of sporangia was highly varia- ble on nutrient media Without giving details of his finding. Tarjot observed that the optimum temperature for fructification ranges from o 25 to 30 C. Medeiros and Alvin (1967) found that a high concentration of carbon dioxide in the atmosphere inhibited the formation of sporangia on infected pods. A high relative humidity (close to 100% R.H.) also had an inhibitory effect. In the absence of carbon dioxide, the most favourable relative humidity for sporangial production was approxima- tely 30 per cent. Light is now generally known to stimulate sporangial production in ~lm~~ (Hemmes snd Hohl, 1973; Clerk. 1974) and it is uni- versally provided when sporulation is deSired. University of Ghana http://ugspace.ug.edu.gh 8 ill MATERIALS AND GENERAL METHODS (i) M!\TERIALS The isolate of p. palmivora used in these studies was obtained from a naturally infected cocoa pod collected from a farm at Aburi, Akwapim. Cocoa pods of the various varieties used in one of the experiments were collected from the Experimental Farms of the Cocoa Research Institute at Tafo. Cocoa pods from which the beans were extracted for raising seedlings for the preparation of root exudates and also, for the preparation of cocoa pod extracts were collected from the Botanical Garden of the University of Ghana. (ii) GENERAL METHODS (8) Mbintenance of Stock Culture Stock cultures of P. palmivora were maintained on slopes of Cassava Dextrose Agar medium (200g cassava: 20g dextrose; 30g agar: 1000 ml distilled water) in McCartney tubes. They were incubated at 250c and sub- cultured at weekly intervals. (b) Production of SporanRia. . on Cocoa Pods Surface sterilized cocoa pods (see page ~ ) were inoculated by making a triangular inCiSion, measuring 1 em along each Side, at the stylar end of each pod and plaCing mycelium bearing agar discs upside down in nd. The pods were then incubated at 250 C in University of Gha9n a http://ugspace.ug.edu.gh sterile rectangular ~erspex containers (22.5 cm long x 16.0 cm in width x 30.5 cm deep) which were open at one end. The pods were placed on moist cotton wool pads and the perspex chambers inverted over them. The mycelium which grew mostly within the husk of the pod sporulated on the surface on emergent hyphal branches. An average ?od of 15 cm long was completely rotted by the fungus in 10 days. (c) Methods of Sterilization McCartney tubes, pipettes, nutrient media and glass distilled water were sterilized by auto~claving for 15 minutes at 1.055 kg/cm2 steam pressure. Cotton wool plugs were temporarily covered with grease paper to prevent the penetration of any condensed water during autOvclaving. Pipettes were also wrapped in grease paper. Petri dishes (5 em and 9 cm diameter) were sterilized by heating at l650 C for 6 hours in an electrically heated oven. Slides and cover slips were thoroughly washed with detergent, rinsed under running tap and in several changes of distilled water and stored in 90 per cent ethanol and flame-sterilized just before use. Inoculating needles and loops were flamed to red-heat and cooled immediately before use. Cocoa pods were thoroughly washed under running tap rface sterilized by washing them with 95 per cent and rinsing thoroughly with sterile de-ionised University of Ghana http1:0// ugspace.ug.edu.gh water. The perspex humidity chambers which held the inoculated cocoa pods were sterilized by washing with 90 per cent ethanol. The inoculating room was always 3prayed with dettol solution (Dettol 5 ml; water 95 ml) 10 minutes immedia- tely before use. (d) Culture of P. palmivora on nutrient media Nutrient agar (15 ml ) in a 9 cm diameter Petri dish was inoculated with .:2 mm culture disc. removed with a No.2 cork borer from the growing edge of a 7 day old culture raised on Cassava Dextrose Agar. The inoculum was placed at the intersection of two diameters. There were four replicates for each treatment. The cultures were incubated initially for four days in dark and then exposed to light of 250 lux for three days. (e) Sporangial germination methods The sporangia were germinated in these studies in liquid media. Suspensions of the sporangia. prepared with either sterile de-ionised water or appropriate medium in sterile Petri dishes (5 cm diameter), each Petri dish holding 10 ml of suspension, were incubated at different temperatures for direct germination (by production of germ tubes) and for indirect germination (by formation of 20OSpores). ures for studies on direct germination of sporan- ._ ___ ________- -=~-~~_=.·~e incubated at 300 c in an fnl'tthat-n ... .AftA .." '_. . ~ & __ University of GhIa".:.n a http://ugspace.ug.edu.gh indirect germination at 22°C provided by an air- conditioned room. (f) Assessment of gro·.7th of culture Growth rate on agar plates was assessed by measuring the diameter of the cultu~e3 along two diameters at right angles to each other at desir~d intervals. (g) Assessment of sporulation The degree of sporulation on the various media in Petri dishes was determined at the end of the incubation period. Four discs 10 mm in diameter, were removed from each culture plate with a No.8 cork borer from four symmetrical pOSitions equidistant from the centre of the culture, 16 rom from the centre of the plate. The mycelium from the four discs of each plate was scraped into 10 ml of sterile distilled '-later in a McCartney tube, and the sporangia detached by stirring with an inoculating needle. The suspension l13S then vigorously shaken for 5 minutes to disperse the sporangia. The number of sporangia per ml of suspension prepared from each plate was determined with a haemacytometer. Each result is the mean of 160 counts from four Petri plates. (h) Assessment of sporangial germination At the end of the desired incubation period, the suspen- sion of sporangia in the Petri dish was examined directly microscope and the germinated and ungerminated of each of the three sporangial length classes University of Gha1n2 a http://ugspace.ug.edu.gh (see Chapter B) in the microscope field, were counted. If observations could not be made at once, a drop of O.lM formal-dehyde was added to each suspension to stop further growth. For assessment of direct germination of the sporangia, any sporangium with a discernible germ tube was counted as having germinated. The definition of germination was thus based on morphology. It is now well known that external evidences of spore germination are long prece- ded by enzymatic changes within the spore, and observa- tions of Hemmes and Hohl (1969) showed that structural changes such as breakdown of the flagella take place in the sporangia of Phytophthora parasitica Dastur long before the emergence of germ tubes during direct germi- na~ion. It is clear that a definition of germination should include this state of physiological change in the spore. Methods are yet to be devised for quick determi- nation of such enzymatic and internal structural changes to suit germination testsof this kind. The numbers of sporangia of each size-class examined are shown in the tables of data. (i) Preparation of Cocoa Pod Extract The husk of a mature medium-sized unripe cocoa pod was removed after the pod had been washed. Ten grams of the husk was ground in 200 ml de-ionised water in a waring .-- - .. - one minute. The thoroughly ground mixture University of Ghana http://ugspace.ug.edu.gh was filtered twice through a Buchrter funnel and Whatman No.2 filter paper until all solid particles were removed from the extract. The extract was stored in a refrigerator until required. (j) Chemicals All chemicals used in the preparation of the media were either of the Annalar grade or of the B.D.H (British Drug House) grade. (k) Culture Media Each 9 em-diameter Petri dish contained 20 ml of agar medium. The compositions of Agar Media used were as follows: 1. Cassava Dextrose Agar: 200g of peeled cassava were boiled in 500 ml of de-ionised water, strained and made up to 1000 ml: Dextrose, 20g; Agar, 20g. 2, Malt-Extract Agar: Malt extract powder, 20g; Agar, 20g; Distilled water, 1000 mI. 3, Glucose-Nitrate Medium: Glucose, 5.4g; NaN03' 1.5g: KH2 P04 , Ig: MgS04.7H20, 0.5g; a-cholesterol. 0.2 mg; Thiamine, 0.5 ml of 1000 ppm stock solution; Agar, 17g; Distilled water, 1000 mI. The composition of this medium was varied to alter both carbohydrate and Nitrogen-compound component for some of the experiments. The details of the modified "e given at the appropriate place in the text. University of Gha1n4 a http://ugspace.ug.edu.gh 4. potato-Dextrose Agar and Yam Dextrose Agar: As in Cassava Dextrose Agar but with potato or yam instead of Cassava. 5. Yeast-Extract Agar: Glucose, 109; Peptone, log. Yeast-extract, 3g; Agar, 20g; Distilled water, 1000 mI. (1) Measurement of pH of M:!dia The pH of the media was measured with a Beckman pH meter. An extra medium in excess of the number of repli- cates required for a treatment was used for measuring the pH after autoelaving. (m) Staining M:!thods The sporangia were stained by the methods outlined below, following the procedure adopted by Hawker, Abbott and Gooday (1968), to investigate the presence and dis- tribution of mitochondria aad RNA, and reserve food sub- stances , lipids, Bnd proteins, in sporangia of different siz-es. Sporangia collected frou the growing edge of the culture on the inoculated pod were transferred onto clean dry glass slides by touching the slides gently with the mycelium-bearing platinum mi.crospatula. The moist s?orangia readily adhered to the slides and were appropriately stained. ~ucleic acid (R.N.A) ':te Sporangia were fixed in acetic alcohol (1 part University of G1h5 ana http://ugspace.ug.edu.gh glacial acetic acid, 3 parts of 95 per cent ethyl alcohol) for approximately 30-60 minutes. The fixed material was brought down to water through a graded series of alcohol and water mixtures. The slides supporting the fixed material were placed in Korson's solution A in a staining dish for 2 minutes (Korson's solution A: 4 per cent orange G in distilled water). The sporangia were washed in distilled water to which a drop of Korson's solution A had been added. It was then placed in Korson's solution B for 15 minutes. (Korson's solution B: 0.15 per cent methyl Green in distilled water). The stain was then drained off and the sporangia stained in Korson's solution C for 5 minutes. (Korson's solution C: 1 part 0.15 per cent methyl Green in distilled water plus 1 part 0.1 per cent Toluidine Blue in distilled water). The sporangia were then rinsed in pure tertiary butyl alcohol and then left overnight in fresh tertiary butyl alcohol to com- plete destaining. The sporangia were then washed in xylol and mounted in Canada Balsam. RNA stained deep blue. 2. Mitochondria The sporangia were stained in 0.01 per cent Janus Green for 30-60 seconds. Mitochondria stained blue. -:!!! ""he presence of lipids was demonstrated uS,1.U2 University of Ghana http://ugspace.ug.edu.gh 16 Sudan III. This stain dissolves in lipid material which then show. 8red colour. The sporangia on the slides were allowed to dry and then a drop of the stain (O.lg Sudan III: 50.0 ml 95 per cent ethyl alcohol; 50.0 ml glycerine) added. The stain was run off the slide after 5 minutes. The sporangia were then mounted in glycerol and examined under the micros- cope. 4. Proteins The sporangia were fixed in acetic-alcohol for 20-30 minutes and then left overnight in 10 per cent perchloric acid at 4°c so as to extract all~. The r~terial was removed the next day, washed twice in distilled water and stained for 30-60 minutes at room temperature in a mixture of equal volumes of 0.1 per cent Safranin and Fast Green. (Mixed equal volumes of stains, allowed to stand for 10 minutes with occaSional shaking, filtered and the ?reci?itate discar~ed). The sporangia were then washed twice in distilled water, washed in two changes of absolute alcohol,cleared in 'xylene and mounted in Canada Balsam. Proteins were stained green. The contents of sporangia of the three different sizes were compared on the basis of intensity of -- _ ,,"" ·, ... ing. University of Gha1n7a http://ugspace.ug.edu.gh Analysis of Sugars by paper Chromatography Sugars present in root exudate of 20 day old cocoa seedlings were detected and their respective quantities estimated by paper chromatography. The solvent system used was iso-Propanol-Water (lPr Aq) (Menzies and Seakins. 1969) made up of 80 ml iso-Propanol and 20 ml de-ionised water. Forty millilitres of the solvent were put inuShandon tUnikit' tank. 1. Application of test solution and subseguent development of the chromatogram The adsorbent used for the development of the chroma- togram was Whatman No.1 chromatographic paper. The test solution was applied as spots along a base line 10 cm from one end of the chromatographic paper strip using a micro- syringe ("Alga" Brand Burroughs, Wellcome and Co., London) calibrated to deliver a volume of 0.01 ml per complete revolution. An amount of 1.0 ml solution was applied at each spot on the paper and delivered at a rate of 0.01 ml at a time. At each delivery, the spot was quickly dried under a current of air from an electric table fan. Each chromatogram was developed for 10 to 11 hours at room temperature of 22 ± 2°C. At the end of this period. the fully developed chromatogram was carefully removed from the chamber, solvent front marked and then hung up to dry freely in the air. at 25°C for four hours. Glucose wss taken as the reference compound and one ~-",uCose solution was run in parallel with the root University of Gha18n a http://ugspace.ug.edu.gh exudate. The total volume of glucose applied at a spot on the paper was 0.05 ml. The glucose served both for identification and rough quantification of the glucose present in the exudate. 2. Detection of SUgars Sugars were detected in developed and thoroughly dried chromatogram by using Alkaline Silver oxide Reagent:- a) AgN03 saturated solution in water 0.1 vol Acetone 20 vol or 100 vol b) NaOH, 0.5g, dissolved in 5 ml water and diluted to 100 ml with ethanol. The paper was first dipped through the silver reagent and the acetone was evaporated. When dry it was dipped through the alkali and again the acetone was evaporated. Dark brown spots began to a:ppear at once on a background which changes through yellow to brown. 3. Identification of developed spots and estimation of concentration of respective substances The sugars were identified by their Rg values, defined as the ratio of the distance travelled by the substances chromatographed and the distance travelled by the glucose spot, and by comparison with known markers. After decisive identification, ap?roximate quantitative determinations were made by visually comparing colour, intensities and sizes of the developed spots with those of ·~s of corresponding markers. University of Ghana http://ugspace.ug.edu.gh 19 (0) Humidity Chambers Solid watch glasses (3.7 x 3.7 x 1.6 cm) with a well 3 cm in diameter and 1.0 cm deep and with transparent glass lids were used as humidity chambers. These served as van Tieghemcells. The well of each watch glass held 2 m1 of the appropriate solution to give the desired relative humidity. The top edge of the watch glass was luted with vaseline in order to form an airtight seal when the lid was placed on it. Sporangia of!. palmivora were dusted oato the centre of the glass lid within a circumscribed area of 1.0 cm in diameter. The glass lid was then gently placed, with the sporangium bearing face downwards onto the watch glass. (p) Maintenance of constant relative humidities To obtain and maintain the different relative humidi- ties except 1001. R.H., sulphuric acid solutions were used according to the data of Solomon (1952) (see Table 1). (q) Statistical AnalysiS Experimental results were statistically analysed where necessary. The least significant difference (L.S.D.) at both 1.0 per cent and 5.0 per cent levels have been quoted. University of Ghana http://ugspace.ug.edu.gh 20 TABLE 1 Aqueous sulphuric acid solutions for maintaining desired constant relative humidities at 25°C. (Extracted from data of Solomon, M,E. 1952) 1- Weight in gm. Weight of water Relative of H2S04 per in gm. per 100g Humidity 100g of solution solution 100 0.0 100.00 95 11.02 88.98 90 17.91 82.09 80 26.79 73.21 70 33.09 66.91 60 38.35 61.65 50 43.10 56.90 40 47.71 52.29 30 52.45 47.55 University of Ghana http://ugspace.ug.edu.gh 21 (r) Experimental precautions 1. Glassware was kept scrupulously clean. Glassware which had been already cleaned with detergents and tap water was rinsed several times with distilled water and allowed to drain before use. 2. Except at the required period of taking results the experiments were not disturbed. 3. The density of sporangial suspension was strictly standardised to 250,000 per millilitre of suspension with the aid of haemacytometer. 4. The incubation chambers covering inoculated cocoa pods were removed for few seconds every morning to let off any gases accumulated in the closed chamber and to wipe off drip?ing condensed water on the inner surface of the chamber. University of Ghana http://ugspace.ug.edu.gh 22' A. RELATIONSHIP BETWEEN THE LENGTHS AND WIDTHS OF SPORANGIA OF P, MLMIVORA There should be a reasonable basis for separation of the sporangia produced by P. palmivora into size classes for any serious study of the relation between size of the sporangia and their functioning or physiology, The typical shape of the sporangium of P. palmivora is ellipsoidal or ovo;·d with the widest part nearer the base. It is papillate and has a short pedicel. Separation of the s?orangia into different size classes could be done by consideration of both the lengths and widths or ratio of length and width (lib), In many instances among fungi, such as species of Cercospora and Cladosporium, where much variation among the spores or polymorphism exists, the lengths have been used as a criterion for separation of the spores because differences in width are normally small or do not exist at all. A cursory glance at the sporangia of ~- ~ during a preliminary examination showed that variation in width was not as prominent as that of the length and a distribution of the sporangia based on lengths alone would be reliable. It was found necessary to confirm this supposition by statistical analYSiS, by calculating the correlation coeffi- cient of lengths and widths and testing for its significance, Sporangia were harvested from the grOWing edge of the area of infection of inoculated cocoa peds and the lengths and ~. :-:'::; n tlorangia, randomly se lee ted, under the micros- , - - -ed using an eye-piece micrometer. The sporangia University of Ghana http://ugspace.ug.edu.gh 23 were transferred into drops of water on slides to keep them turgid during measurement. The data obtained were used to draw a linear scattergram shown in Fig.1, and the correlation coefficient (r) calculated using Bravais - Pearson Product-Moment Method. The correlation coefficient (r) was +0.47. To check the significance of r, the standard formula 1 _ r2 a (r) = '-N- was used, where a is the standard error of the coefficient of correlation found in the sample distribution, and N is the total number of cases in the sample. The value of a (r) was found to be 0,20 and this is not significant at both 1% and 5% level. This result therefore shows that the widths of the spora- ngia did not vary with their lengths, and widths being quite close, the length alone was a sufficient criterion for distin- guishing between them. The length of the spore alone has been useful in other pertinent studies. For example, Welles (192'), investigating the effect of environmental conditions on the dimensions of conidia of members of the Genus Cercospora demonstrated that the length was dramatically altered whilst very little change occurred in the width of spores. He assessed the effect of the 7S by changes in conidial length. University of Ghana http://ugspace.ug.edu.gh 24 100 90 80 ~ . ... . ~ .. :.I.:. · C) .. .. z . UJ • . ..J ·.. . ..J G'" ... ·.. z 30 a'": . 0 .. :••. . • a.. 20 III 10 o 10 20 30 40 so SPORAr"GJAL WIDTH IN Mm ~ IG.I SCATTER GRAM SHOWING LINEAR CORRELATION TENDENCY BETWEEN THE LENGTHS AND WIDTHS OF SPORAN- GIA OF e PALMIVORA FORMED ON AMELONADO COCOA PODS. University of Ghana25 http://ugspace.ug.edu.gh B. ~ORI?!,TIONOF THE SPORANGIA INTO SIZE-CLASSES The sporangia of P. palmivora vary considerably in size. Some of the sporangia obtained from infected Ame10nado cocoa pods almost do not show any difference between length and width (see Fig.2), and are between 12.5 and 15.5 ~ long. The longest are 57.5 to 60.5 \J m long. Between these extreme forms is a range of size of sporangia of any possible length. The sporan- gia needed to be separated into recognisable size-classes based on sporangia1 lengths to be adopted throughout these studies. Sporangia from four inoculated cocoa pods were gently removed with a microspatula and shaken in distilled water in a MCCartney tube. The sporangia usually dispersed very readily but settled quickly and the suspension was shaken when sample drops of the suspension were to be taken to bring all sporangia into suspension. Drops of the suspension were put on slides and observed under the microscope without putting on cover slips. The lengths of 500 randomly selected sporangia taken from not less than 20 suspension drops were measured USing an eye-piece micrometer. Fig.2 is a sample of a collection of the sporangia depicting different sporangial sizes and Plate 1 gives details of variations in shape which might not be represented in Fig.2. The data in Table 2 show the frequency of the sporangia in the various length-classes constructed at 3.0 ~m intervals. The same data are shown in Fig.3 as histograms for clarity. The lengths of the sporangia ranged from 12.5 to 60.5 um which could to three size-classes, determined by the nature of the frequencies of the sporangia in the length- University of Ghana http://ugspace.ug.edu.gh 26 classes. On the basis of length, "Small" sporangia fell between the range 12.5 and 24.5 um. Those designated "Large" sporangia were more than 48.5 u m long and sporangia with lengths between these two extremes were placed in the "Med:lUmn size- class. The respective frequencies of the sporangia formed on the Amelonado cocoa pod in the three size-classes - small, median and large - were 8.7, 87,1 and 4.2 per cent. University of Ghana http://ugspace.ug.edu.gh 27 CX?oo8oo$ Q ..... . ······ 0 ·· ··················, I I SO,(Jm FIG.2 SPORANGIA OF PHYTOPHTHORA PALMIVORA SHOWING VARIATION IN SIZE o .. SMALL SIZED; b. MEDI\lfIC SIZED; c. LARGE SIZED. University of Ghana http://ugspace.ug.edu.gh 28 PLATE 1. Photomicrograph of sporangia of P. palmivora showing both differences in size and sha?E of the sporangia. x 110. University of Ghana http://ugspace.ug.edu.gh 29 TABLE 2 Frequency distribution of lengths of sporangia of P. pa1mivora formed on AmeJonado cocoa pods at 25°c in an atmosphere of 100 ?er cent R.H. Percentage of Length-class No. of sporangia in in um sporangia length-class 9.5 -12.5 0 0.0 12.5 -15.5 8 1.6 15.5 -18.5 2 0.4 18.5 - 21.5 23 4.8 21.5 - 24.5 9 1.9 24.5 -27.5 21 4.4 27.5 - 30.5 64 13.4 30.5 - 33.5 59 12.8 33.5 -36.5 61 12.9 36.5 - 39.5 42 8.9 39.5 - 42,5 63 13.3 University of Ghana http://ugspace.ug.edu.gh 30 tABLE 2 cont'd. Frequency distribution of lengths of sporangia of P. pa1mivora formed on Ame10nado cocoa pods at 25°C in an atmosphere of 100 per cent R.H. Percentage of Length-class No. of s~rangia in in urn sporanBia length-class 42.5 - 45.5 50 10.6 45.5 -48.5 6 1.3 48.5 - 51.5 45 9.5 51.5 - 54.5 8 1.6 54.5 - 57.5 10 2.0 57.5 - 60.5 3 0.6 60.5 - 63.5 0 0.0 University of Ghana http://ugspace.ug.edu.gh 1 : 9· 5 - 12' 5 '-1m 2:12'5-15'5 um 3:15-5- 18' 5 #Jm 4:18'5-21- 5 Jim 5:21'5-24-5 '-1m 6 :24-5-27,5 Jim 7 :27-5-30-5 Jim 8:30'5-33 ' S #Jm 9 :33- 5- 36- 5 '-1m 10:36'5-39-5 #Jm II :39-S-42-5' '-Im 12 :42' 5-45' 5 #Jm r-4 13 :45 ~-48'S #Jm C"l 14 :48 '5-51 - 5 '-1m 15 51 '5-54 'S#Jm 30 16 S4 '5-57-5#Jm > u 17 57 'S-60'S#Jm z 18 6Q'S-63-SIJIYl UJ 20 :::> o UJ ex: ~.. 10 o 0' '===' ~ t=== I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 CLASS-LENGTHS FIG.3 DISTRIBUTION OF CLASS-LENGTHS OF SPORANGIA OF P. PALMIVORA FORMED BY MYCELIUM GROWING ON AMELONADO COCOA POD AT 2S o C . University of Ghana htt3p2 ://ugspace.ug.edu.gh c. ~IZE OF SPORANGIA OF P. PALM IV ORA FORMED ON DIFFERENT NUTRIENT MEDIA The genus Phytophthora is composed of some highly plasttc fungi. M$ny morphologicsl as well as physiological characteris- tics of these organisms are generated to a great extent by the environmental conditions. The experiments described in this chapter and the next extend existing pertinent knowledge on P. palmivors. Waterhouse (1974) reported that the size of ~ palmivora sporangia vary widely according to medium, host, age of culture, moisture and light. The way in which each influen- ced the size of the sporangia was however not stated. The influence of the environment on the sporangia of other Phytophthora species is very clearly defined. The observations of Leonian (1927) still remain one of the best illustrations, and have been described in detail in the Introduction. Leonian inoculated green banana fruits, fruits of egg-plant, lemon, orange, pepper and tomato and tubers of potato with Phytophthora caspici, Phytophthora mexicana and Phytophthora In spite of the ready infection of all the hosts, the fungi produced sporangia in great abundance only on egg plants, peppers and tomatoes. There were in addition striking differen- ces in the morphological features, their Size, shape and nature of the papillae, among sporangia formed on the different host plants. Sporangia formed by all three species on tomatoes were and very large sporangia were formed by ~ .. mexicana on pepper and by ~o~ivors on e~~ ------------~ University of Ghana http://ugspace.ug.edu.gh 33 plant. The growth rate, the degree of sporangium formation and morphology of the sporangia of P. palmivora on four natural nutrient agar media were investigated. The media used were Cassava.Dextrose agar, ~lt-Dextrose agar, Potato-Dextrose agar and Yam-Dextrose agar. Inoculated Petri plates, five replicates for each medium, were incubated at 2Soc. The plates were kept in darkness for the first four days and then exposed to light of 250 lux for the remaining days. The diameters of the colonies were measured daily. beginning on the 4th day of incubation and the amount of sporangia produced estimated on the 8th day. Finally, the lengths of randomly selected sporan- gia from each culture plate were measured to assess the frequen- cy of sporangia of the three size-classes. The data provided therefore represent samples from all plates of each treatment and for each treatment not less than 900 sporangia were measured. The results are presented in Tables 3 and 4, and the proportions of the sporangia of different size-classes formed on these media are com~red with those on cocoa pod in Fig.4. It was observed that Cassava-Dextrose agar was the best medium for growth. Whilst the culture on this medium covered the plate in seven days, cultures on Malt-Dextrose agar which supported the Slowest vegetative growth had a mean diameter of 66.8 mm at the end of the incubation period (Table 3). When the denSities of sporangia in suspensions prepared the culture plates, according to the method ---e 11, were calculated, it was found that University of Ghana http://ugspace.ug.edu.gh 34 TABLE 3 Growth of Pc palmivora inoculated on different media and incubated at 25°C in darkness initially for 4 days and then in light of 250 lux for 3 days. ----------- .~----------------------------------------------------- Me an Co 1 ony Dia me t er a ft er i ne li cat ec l days of incubation Agar Medium 4 5 6 7 Cassava- Dextrose 64.4 ± 1.6 78.6 ± 0.8 83.1 ± 1.0 89.8± 0.3 Malt- Dextrose 38.9 ± 1.5 39.8 ± 1.7 59.8 ± 2.3 66.9 ± 3.4 I Potato- Dextrose 40.2 ± 1.0 48.7 ± 1.3 I 58.1 ± I 0.4 71.8 ± 0.6 Yam- Dextrose 50.1 ± 0.5 61.2 ± 0.7 I 72.8 ± 0.7 .. 78 .7 ± 1.0 ----L I University of Ghana http://ugspace.ug.edu.gh 35 Cassava-Dextrose agar was again the most suitable medium for sporulation. P. palrnivora sporulated very poorly on ~lt­ Dextrose and Yam-Dextrose agar. The calculated densities of the suspending medium prepared from cassava-dextrose, Malt-dextrose, 3 Potato-dextrose and Yam-dextrose agar were 40 x 103, 4 x 10 , 4 x 103 and 36 x 103 sporangia per ml, respectively. Differences in the effect of the media were also detected in the size of the sporangia. The results in Table 4 clearly show that there were more numerous small-sporangia (less than 24.5 ~m long), 61.2 per cent, on the Cassava-dextrose plates than on the others where the frequencies ranged from 16.3 to 29.8 per cent. The Size-class with the largest percentage of sporangia in the three other media was the Mediwm size-class. Potato-dextrose agar showed the highest value of 79.1 per cent and yam-d~xtrose agar, the least, 68.2 per cent. 2 A X analysis (Table 4e) showed that the differences in the pattern of distribution of the sporangia of each population into the three size-classes was significant at both 11. and 5% levels. University of Ghana http://ugspace.ug.edu.gh 36 TABLE 4a Distribution of sporangia of ~lmivora formed by myceliuo growing on Cassava-Dextrose Agar at 250 C. (Cultures incubated initially in darkness for four days and then in light of 250 lux for three days) Sporangial size- Percentage of class based on No. of sporangia in sporangial length sporan~a size-class Small 705 61.2 « 24.5 ~m) Medi~m 427 37.0 (24.5 -48.5 \.fIIl) Large 21 1.8 (> 48.5 ~m) University of Ghana http://ugspace.ug.edu.gh 37 Distribution of sporangia on ~l~v~ formed at mycelium growing on Mnlt-Dextrose Agar by 250 c. (Cultures incubated initially for 4 days in darkness and then in light of 250 lux for 3 days) Sporangial size- Percentage of class based on No. of sporangia in sporangial length sporangia size-class Small 286 27.9 « 24.5 ~m) MedUtro 738 71.9 (24.5 - 48.5 lJm) Large .2 0.2 (> 48.5 lJm) University of Ghana http://ugspace.ug.edu.gh 38 TABLE 4c Distribution of sporangia of P. ?almivora formed by mycelium growing on Potato-Dextrose Agar at 25°C. (Cultures incubated initially in darkness for 4 days and then in light of 250 lux for 3 days) Sporangial size- Percentage of class based on No. of sporangia in sporenBlel length sporan~g~i~a ______~ s~i~z~e~-~c~l~a~s~s _______ __ Small 162 16.3 ( < 24.5 lJIl) Medium 785 79.1 (24.5 -48.5 ~m) Large 46 4.6 (> 48.5 llm) ------------------- University of Ghana http://ugspace.ug.edu.gh 39 TABLE 4d Distribution of sporangia of P. palmivora formed by mycelium growing on Yam-Dextrose ASar at 250c. (Cultures incubated initially in darkness for 4 days and then in light of 250 lux for 3 days Sporangial size- Percentage of class basec on No. of sporangia 1n ___s -22,rangial length sporangia size-class Small 303 29.8 ( < 24.5 ~m) Medi\lfl) 692 68.2 (24.5 - 48.5 ~) Large 20 2.0 (> 48.5 ~m) ------------------_._--- University of Ghana http://ugspace.ug.edu.gh TABLE 4e x2 Analysis of the data on sporaneial size (Tables 48 - d) obtained by cultur.ine P. palmivora on different natural media. Total Number of Sporangia in size-class Culture Medium Small Medium Large «24.51J m) (24.5 -48.5pm) ( 48.5lJm) Cassava-dextrose agar 705 427 21 I 1153 Malt-dextrose agar 286 738 2 1026 Potato-dextrose I agar I 162 785 46 993 Yam-dextrose I near I 303 692 20 1015 I 1456 2642 89 4187 x~ 577.3 at 5% level Table value 2 ~6 12.6 at 5% (p <0.001). University of Ghana4 1h ttp://ugspace.ug.edu.gh 100 tz 80 ILl :::l 60 0 COCOA POD ILl a: 40 10. ~ 20 0 0 )0- 100 ffi 80 :::l CASSAVA- 0 60 DEXTROSE aw: 40 AGAR 10. ~ 20 0 O . 100 tz 80 W MALT-DEXTROSE :::l 60 AGAR 0w 0: ' 40 II.. ~ 20 0 0 100 >- ~ 80 POTATO- w 60 DEXTROSE :::l 0 AGAR w a: 40 u.. 20 ~ 0 0 100 )0- U Z 80 w :::l 60 YAM-DEXTROSE 0w AGAR 0: 40 10. ~ 20 0 0 FIG . 4 DISTRIBUTION OF CLASS-lENGTHS OF SPORANGIA OF RPAlMIVORA FORMED BY MYCELIUM GROWING ON DIFFERENT AGAR MEDIA AT 25°C . It 5·0-15·9,4.Lm 2: 16·0-26 ·9,4.Lm 3:27·0-37 ·9pm . 4 : 38 '0-48 '9pm 5 : 49 ·0-59 ·9,4.Lm 6·.60 ·0-71 ·9~ . University of Ghana http://ugspace.ug.edu.gh 42 D. INFLUEOCE OF CARBOHYDRATES AND NITROGEN COMPOUNDS ON S PORANGIAL SIZE The striking modification of the frequencies of sporangia of different sizes by Cassava-dextrose agar was undoubtedly due to the nature of some of its component compounds. It will not be possible to examine the effect of every compound that has been identified in the tuber, but by selecting a few carbohy- drates and nitrogenous compounds, an information could be obtained on the role of at least two important groups of the components. This does not naturally exclude the ?ossibility of any role that other compounds such as growth substances, mineral salts and toxic compounds (e.g., cyanide) might play. P. palmivora was grown on different agar media at 250c to examine the effect of the follOWing on the morphology of the sporangia: (a) different concentrations of glucose (b) different concentrations of sodium nitrate (c) different sugars (d) different nitrogen compounds. The Glucose-Nitrate Medium adopted by Hendrix (1965), which contained cholestrol and which Hendrix found to be very suitable for sporulation of six pythium species and 21 Phytophthora s?€cies, including P. palmtvora, was used With the necessary modification for the series of investigations which have been outlined. _~nt concentrations of glUcose and sodium nitrate - - !nts (8) and (b) are shown in the tables of University of Ghana http://ugspace.ug.edu.gh 43 results (see Tables 5 to 8). The effects of three sugars, Fructose, Galactose and Sucrose, were studied using a single concentration of 5.4 g/t. Ammonium tartf.te, Asparagine and Pe?tone, each at a concentration of 1.5 g/2, replaced sodium nitrate in the last series of experiments which investigated the effects of other nitrogenous compounds beside sodium nitrate. The cultures were incubated, as already described, in dark- ness initially for four days and then exposed to light for the subsequent three days. The growth rate on the various media was determined by colony diameter measurements on the 4th to 7th days. At the end of the incubation period, the degree of sporulation was assessed, and the lengths of 1000 randomly selected sporangia for each treatment were measured. The results are ?resented in Tables 5 to 12. Growth Rate P. palmivora grew at the same rate on media containing different concentrations (1.0 to 5.4 g/t) of Glucose, and which was faster than the rate on any of the other three sugars. Growth on Galactose and Sucrose media was just slightly inferior and the finsl mean diameters were 85.S and ~7.9 mm, respectively, in comparison with a mean diameter of 90.0 mm on the Clucose medium. Growth on llructose medium was considerably slower and the fins1 mean colony diameter was 74.4 mm (Tables 5 and 9). There was seemingly no significant difference among the he colonies on media containing the four Nitro- Mycelium on the Sodium nitrate medium (NaNOJ University of Ghana http://ugspace.ug.edu.gh 44 at 1,5 g/i), however, had a faster initial growth rate noticea- ble up to the 5th day (Tables 7 and 11). A concentration of 1.0 glt sodium nitrate seemed to be optimal for growth of ~ palmivora (Table 7). The culture grew faster on this medium and covered the plate on the 6th day, a day earlier than the rest which did so on the 7th day. Degree of Sporulation The extent to which the media supported sporulation - sporangial formation - varied considerably. Among the sugars tested, Glucose was found to be the best ~dium for sporulation and Galactose the poorest. Plates 2 to 5 show typical views of sporangia on plates containing different glucose concentrations. Since the results are not included in the tables of data, the exact values obtained are presented here: 3 Glucose at 1.0 g/t 60 x 10 sporangia Iml. Glucose at 2.0 g/t 68 x 103 sporangia Iml. Glucose at 3.0 g/t 64 x 103 sporangia Iml. Glucose at 4.0 g/t 60x 103 sporangia 1m!. Glucose at 5 .. 0 g/i 60 x 103 sporangia 1m!. Fructose at 5.4 g/£ 12 x 103 sporangia Iml. Galactose at 5.4 g/£ 8 x 3 10 sporangia Iml. Sucrose at 5.4 g/t 24 x 103 sporangia Iml. Sodium nitrate at the concentration (1.5 g/t) at which its effect could be c om pa red t t h 0 at of At!IIllOniUIII tartrate, Aspara- was strikingly the best medium for sporangial University of Ghana http://ugspace.ug.edu.gh 45 quantity eight times gre~ter than the production, giving a least suitable, ADIllOnium tartrate. ~e full data are as follows f Sodium nitrate at 0.25 g/t 44 x 10 3 sporangia Im1. 3 Sodium nitrate at 0.5 gIl 64 x 10 sporangia Im1. 3 Sodium nitrate at 1.0 glR. 72 x 10 sporangia Im1. 3 Sodium nitrate at 1.5 g/t 128 x 10 sporangia Iml. Ammonium tartrate at 1.5 glR. 16 x 103 sporangia Im1. 3 Asparagine at 1.5 g/t 76 x 10 sporangia Iml. Peptone at 1.5 glR. 40 x 10 3 sporangia Iml. Sizes of Sporangia Different concentrations of either Glucose (Table 6) or Sodium nitrate (Table 8) supported formation of sporangia of the different sizes in almost the same proportion. Dramatic effects were recognised when these were compared with the patterns on media containing the other sugars and Nitrogenous compounds. Thus Sucrose (Table 10) and Asparagine (Table 12) encouraged the formation of large sporangia. The proportions of the sporangia which fell under the category of "large sporangia" were 31.5 and 47.1 per cent , respectively, whilst the highest in the rest was 18.3 ?er cent on the Glucose medium. Ammonium tartrate (Table 12) and Fructose (Table 10) on the other hand, were media for production of small-sized sporan- gia. where 12.0 and 23.3 per cent of the sporangia were found to . -: =-=..! 1l1 size-class. University of Ghana http://ugspace.ug.edu.gh 46 TABLE 5 Growth of P. palmivora inoculated on Glucose-Nitrate Medium containing 0 different concentrations of Glucose and incubated at 25 C 1n darkness initially for 4 days and then in light of 250 lux for 3 days. Mean Colony Diameter (mm) after indicated days of incubation Glucose Concentra tion --------- (g/ ~) 4 5 6 7 1.0 72.5 ± 1.9 I I I 74.0 ± 0.6 86.2 ± 5.6 90.0 ± 0.0 2.0 69.0 ± 5.4 I 79.8 ± 5.6 90.0 ± 0.0 90.0 ± 0.0 3.0 75.0 ± 1.3 83.0 ± 2.6 90.0 ± 0.0 90.0 ± 0.0 4.0 72.0 ± 3.5 84.0 ± 0.2 89.9 ± 0.1 89.9 ± 0.1 5.4 72.8 ±3.l 83.3 ± 2.6 I 90.0 ± 0.0 90.0 ± 0.0 University of Ghana http://ugspace.ug.edu.gh 47 TABLE 6 Percentage of sporangia of P. palmivora formed by mycelium growing on Glucose-Nitrate Medium containing different concentrations of Glucose at 25 0 C among sporangial size-classes. (Cultures incubated initially in darkness for 4 days and then in light of 250 lux for 3 days) Percentage of Sporangia in size-class Glucose based on sporangial length Concentra tion (g/9.) SlII!lll I Medium Large « 24.5 1-1 m) I (24.5 -48.5JJD) I ( > 48.5 1Jm) ---- I 1.0 3.U i I I 79.7 I 16.5 2.0 2.3 I 79.2 I 18.5 3.0 2.4 82.7 14.9 4.0 4.3 85.3 5.4 4.0 77.2 J 10.4 18.8 University of Ghana http://ugspace.ug.edu.gh 48 TABLE 7 Growth of R. palmivora inoculated on Glucose-Nitrate Medium containing 0 different concentrations of Sodium-nitrate and incubated at 25 C in darkness initially for 4 days and then in light Of 250 lux for 3 days. - --- MeSn Colony Diameter (mm) after indicated Sodiwn- days of incubation Nitrate Concentra- tion (g/9.) 4 5 6 7 0.25 56.8 ± 2.1 71.8 ± 1.3 84.4 ± 3.4 90.0 ± 0.0 0.5 57.6 ± 4.3 80.6 ± 3.4 88.8 ± 1.4 I 90.0 ± 0.0 1.0 64.9 ± 3.4 83.8 ± 2.6 90.0 ± 0.0 I 90.0 ± 0.0 1.5 63.9 ± 6.2 82.0 ± 2.2 I %.4 ± 4.5 loooo ± 0.0 - . University of Ghana http://ugspace.ug.edu.gh 49 TABLE 8 Percentage of sporangia of P. palmivor~ formed by mycelium growing on Glucose-Nitrate Medium containing different concentrations of Sodium- Nitrate at 250c among sporangial size-class. (Cultures incubated initially 1n darkness for 4 days and then in light of 250 lux for 3 days) Percentage of sporangia in size-class Sodium (size based on sporangia1 length) Nitrate Concentration (sIt) Small Medium , Larse ( < 24.5 ~m) (24.5 - 48.5 ~m) (> 48.5 \.1m) -----------------~-------------~-------------+-------------- 0.25 1.5 86.5 12.0 0.5 1.2 88.2 10.6 1.0 1.1 85.9 13.0 1,5 4.6 85.7 9.7 University of Ghana http://ugspace.ug.edu.gh 50 TABLE 9 Growth of P. palmivor8 inoculated on Agar Nutrient Medium containing different Carbohydrates a8 C-source and incubated at 25°c in darkness initially and then in light of 250 lux for 3 days. ----- [ Mean Colony Diameter (mm) after indicated days of incubation Carbohy- drate (5.4 glR,) 4 5 1 6 7 Fructose 49.2 ± 5.6 51.9 ± 8.6 I 61.6 ± 11.5 74.4 ± 3.9 Galactose 58.8 ± 3.2 69.7 ± 3.2 I 80.6 ± 3.2 85.5 ± 4.1 Sucrose 57.0 ± 5.7 69.5 ± 7.3 82.0 ± 8.9 87.9 ± 2.4 ----- University of Ghana http://ugspace.ug.edu.gh 51 TABLE 10 Percentage of sporangia of P. pal~ formed by mycelium growing on A8ar Nutrient Medium containing different carbohydrates as C-source and incubated at 25°C among sporangial size-classes. (Cultures incubated initially in darkness for 4 days and then in light of 250 lux for 3 days) Percentage of sporangia in size-class (size based on sporan8ial length) Carbohydrate (5.4 g/i) Small Mediam I Larre ( <24.5 lJm) (24.5 - 48.5 lJm) I (> 48.5 l1m) I I Fructose 12.0 75.4 I 12.6 Galactose 1.7 97.9 0.4 Sucrose 0.5 68.0 I 31.5 University of Ghana http://ugspace.ug.edu.gh 52 TABLE 11 Growth of P. palmivora inoculated on Agar Nutrient Medium containing different Nitrogen compounds as N-source and incubated at 25~ in darkness initially and then in light of 250 lux for 3 days. University of Ghana http://ugspace.ug.edu.gh S3 TABLE 12 Percentage of sporaneia of ~ plllmi~ formed by mycelium growing on Agar Nutrient Medium containing different nitrogen compounds as N-80urce and incubated at 2SoC amone sporangial size-classes. Percentage of sporangia in size-class (size based on sporangial length) Nitrogen Compound (1.5 g/t) Small Mediom Large ( <24.5 \.1m) (24.5 - 48.5 lim) ( >48.5 lim) ---- I A!II!IOnium tartrate I 23.3 74.5 2.2 AspaTsgine 0.4 52.5 47.1 Peptone 0.4 I 93.3 6.3 University of Ghana http://ugspace.ug.edu.gh 54 PLATE 2. Photomicrograph of sporangia of P. palmivora of a typical section of sporulating culture growing on a Glucose-Sodium nitrate agar medium containing 2.0 B/~ Glucose. x 45 University of Ghana http://ugspace.ug.edu.gh 55 PlATE 3. tbotomcrograph of sporangia of P. palmivora of a typical section of sporulating culture growing on a Glucose-Sodium nitrate agar medium containing 3.0 g/r;. Glucose. x 45 University of Ghana http://ugspace.ug.edu.gh S6 . '.. .~, ~ ~ .',,, 'l" .. ,' \ . ..,...... :;'.' ~'' ~ '# ,• ' '.'f'-ll.t - ,... t ....' ,' 'J)l';.'.' ~, .' ... ;;.\ ,.~ -,' . ~~ .'4 V ' , I. ~.. ~~~ . f.' ..' "',~,- • , ,.~. .~ .;.,.' - PLATE 4. Photomicrograph of sporangia of P. palmivora of a typical section of sporulating culture growing on a Glucose-Sodium nitrate agar medium containing 4.0 g/l Glucose. x 45. University of Ghana http://ugspace.ug.edu.gh 57 PlATE 5. Photomicrogra?h of sporangia of P. palm1vora of a typical section of sporulating culture on a Glucose-Sodium nitrate medium containing 5.4 g/i Glucose. x 45. Note the similarity in degree of sporulation on media containing the different concentra- tions of Glucose. University of Ghana http://ugspace.ug.edu.gh 58 E. SIZE OF SPORANGIA OF P. PALM1VORA FORMED ON PODS OF DIFFER.Em' COCM CLONES The morphology of s1,lOrangi8 of ualtniVora formed on pods of different clones of cocoa was next investigated. It is known from field oblerv8tions, records of the natural incidence of the disease, and more recently from artificial tests, that individual cocoa trees in different areas show different levels of suscepti- bility. This fact has encouraged workers in various research institutes to search for resistant material in both cultivated cocoa and in wild trees growing in the forest. These efforts have yielded an impressive list of cocoa clones with some degree of resistance to P. palmivora. Some of the clones with 8 satis- factory level of resistance that have been employed in tests at the Cocoa Research Institute at Tafo, Ghana, are the Trinitario varieties 527, V6. Acu 85, ¥44 and D70 (Wharton 1960, 1961, 1962; Asomaning 1964), T~/799 (resultant clone from cross between the Amazon varieties IMC 60 and Na 34) (Asoenning, 1964) and T79/50l (clone from cross between the Amazon varieties Na 32 and Pa 7) (Asomaning, 1964). Several other tests have been reported from almost all cocoa growing countries, and breeding programmes are still continuing in attempts to produce yet more resistaatclones. It will be difficult to obtain a totally resistant cultivar hence the search for resistant material should be linked with other control measures. If sporangia of different sizes possess different physiological characteristics. and if cocoa clones - .".-- of sporangia they produce, the lDIlnner in which University of Ghana http://ugspace.ug.edu.gh 59 the clon~B influence sporangial si~e is an important information. An examination of the sporangia on pods of four cloneB, Amelonado, T85/799, T63/967 and T79/467 was made. The choice of clones among thoBe available was determined by availability of mature pods at the time of testing. Inoculated pods were incubated at 2SoC in the humid chambers. On the 5th day, samples of sporangia were mounted in distilled water and their lengths measured. There were eight pods for each variety and the sporangia measured, not less than a thousand for each variety came from all the inoculated pods. The frequencies of the sporangia in the three size-classes are shown in Tables 13 - 16. There was a greater proportion of "Large" sporangia, 26.6- 36.1 per cent, in sporangial populations of the three clones than that of Amelonado (20.0 ?er cent). Consequently, the percentage of "Small" and "Medium-sized" Bporangia fell below those for Amelonado in each caBe. In all, however, the median- sized sporangia were the most frequent. A x2 analysiB showed that the differences in the pattern of distribution of the Bporangia of each population into the three Bize-classes waB Bignificant at both 1% and 51. levels. The relevant information iB provided in Table 17. University of Ghana http://ugspace.ug.edu.gh 60 TABLE 13 Distribution of sporangia of ~. palmivora formed by mycelium growing on Amelonado cocoa pods at 25°c among the sporangial size-classes. ---------------- Sporangial size- Percentage of c lass based on No. of sporangia in __ ~!!.ngi8l length_ s120ransia size-class Small 102 9.1 ( < 24.5 lJ m) Medium 794 70.9 (24.5 -43.5 lJm) Large 224 20.0 (> 48.5 lJIIl) University of Ghana http://ugspace.ug.edu.gh 61 TABLE 14 Distribution of sporangia of P. pa1mivora formecl by mycelium growing on variety T85/799 cocoa pocls at 25°C among the sporangial size-classes. Sporangial size- Percentage of class basecl on No. of sporangia in __. ...!.22rangial length spora~ __. ....;s~i::z:.;:e~-.:c;.:l=a::.s::.s __. Small 98 7.9 « 24.5 um) Mecllum 766 61.3 (24.5 - 48.5 \.1m) Large 384 30.8 (>48.5 \.1m) - ---------- University of Ghana http://ugspace.ug.edu.gh 62 TABLE 15 Distribution of sporangia of P. palmivora formed by mycelium growing on variety 163/967 cocoa pods at 25°c among the sporangial size-classes. Sporangia1 size- Percentage of class based on No. of sporangia in sEoranSial length .!E. 48.5 jJm) University of Ghana http://ugspace.ug.edu.gh 63 TABLE 16 Distribution of sporangia of P. palmivora formed by mycelium growing on variety T79/467 cocoa pods at 250C among the sporangial s he -classes. Sporangial size- Percentage of class based on No. of sporangia in sporansial lensth s~ransia size-class Small 70 5.4 « 24.5 llm) Medium 759 58.5 (24,5 - 48. 5 um) Large 468 36.1 (> 48.5 \.lm) University of Ghana http://ugspace.ug.edu.gh 64 ~l ..;. Analysis of the data on sporaneial size (Tables 13 - 16) obtained by culturing P. palmivora on different cocoa clones. '--- Total Number of Sporangia in size-class Cocoa Variety T Small ~1edillm Large « 24.5 lJ m) (24.5 - 48.5 \J m) (>48.5 lJm) I Amelonado 102 794 I 224 1120 T85/799 I 98 766 I 384 1248 T63/967 98 93(, 374 1408 T79/467 70 959 468 1297 368 3255 468 5073 2 Y6 a 33.3 at 5% level Table value X! ~ 12.6 at 5% (P < 0.001) University of Ghana http://ugspace.ug.edu.gh 65 F. POSITION OF SPORANGIA OF DIFFERENI SIZES ON SPORUlATING HYPHAL BRANCH A specific sequence in the formation of the different morpho- logical types in the chain of polymorphic or dimorphic spores has been recognised in some fungal species. Cladosporium harknessii (Peck) comb. nov. (= ~ harkness!i Peck) conidia at the upper section of the conidial chains, hence, the first formed spores are short, while towards the base of the chains the coni- dia are larger and almost cylindrical (Hughes, 1953). In~­ ~ ~ Link ex Fries (Ingold, 1956) and Leveillula ~ (Lev.) Arn. (Clerk and Ayesu-Offei, 1967) there are two types of conidia; 1n·~ each case the first conidium. in the chain is different from the succeeding ones. Although the mode of sporulation by P. palmivora has often been described, the posi- tion of the sporangia of different sizes has not been defined. Waterhouse (1974) noted that the sporangiophores are narrower than the aerial hyphae and bear no swellings. Growth is sympodisl, the tip forming a sporangium, and a branch from immediately below then continues growth so that the sporangium. has only a very short stalk. As many as 20 sporangia may form in a sympodium in this way under optimum conditions, though usually fewer. An experiment was designed to find out whether a distiuct pattern of distribution of sporangia of different sizes occur in the sympodium. Sterile glass cylinders 1 em long and 1.5 cm in diameter a separate sterile Petri dish and 20 ml of University of Ghana http://ugspace.ug.edu.gh 66 melted Potato-dextrose agar poured into the Petri dish, and the glass cylinders adjusted to a central position before the agar solidified. Meanwhile, tiny solidified Potato-dextrose agar discs, 2 rom in diameter and 2 mm thick. were placed in the centre of sterile g14ss slides and inoculated with minute frag- ments of hyphae of p~ palmivora. The slides with their loads of inoculated micro-agar discs were next placed, facing upwards, on the glass cylinders standing in the Petri dishes and the o dishes closed. The whole set was incubated at 2S C for 6 .ays. The Potato-dextrose agar in the Petri dish maintained a congenial atmospheric hu~idity for growth of the micro-culture on the slide. The hyphae of the micro-culture grew out from the agar and ran along the surface of the glass slide. sporulating on the glass surface. This provided an opportunity of studying sporu- lating branches completely undistu~bed and retaining the extremclM caducuous sporangia in position. Measurements of the lengths of the sporangia on several sympodia were made, noting at the same time the position of each in the cluster. Table 18 shows all the pertinent data; the number of clusters examined, the number of sporangia measured, and the percentage of sporangia of a particular size (small, medigm and large) in the respective positions in the cluster. Clearly a sporangiophore produced mostly large sporangia and mediu~sized sporangia at the initial stages of sporulation g.S). Larger sporangia were commonly found University of Ghana http://ugspace.ug.edu.gh 67 at the basal part of the sympodium and smaller ones at the distal end. The exact distribution 1s shown in Table 17. The camera lucida drawings in Fig.5 were made to show not only size of the sporangium in relation to pOSition of the sporangium in the sympodium, but also to portray differences in the length of the stalk of the sporangium, and the variation in distances between a sporangium and the succeeding one. University of Ghana http://ugspace.ug.edu.gh 58 ~ Sin Ilength) of mature sporangia of p palmlyora in relation to position on sporulating hypha of mycelium growing on Cassava Dextrose Agar at 25°C. (5, < 24'S,um i M, 2"·S-48·S,um; L,>48-S,um) Position Mean Distribution of in sporan- No-o' length 0' closs lengths 9101 cluster sporangia sporangium of ('rom bose) \n ,um sporangia (Ofo FREQUENCY) 100- 50 4S-0t 6-0 50- FL, 010-0~ SO 40-6±S'7 '::R 2-0 4'0 0 3 50 3S-4± 5-3 '::R o 6-0 0-0 100- 4 46 28-6± 6- 6 SO~ - O~O-O, 100- 42 27·6±6·3 5or"R o _2_8_-6~_~ 0'0 ' 100- 6 22 213±4 -0 50F=b 0 ___2 _B_'_6~ 0-0 5' M L University of Ghana http://ugspace.ug.edu.gh 69 ,, / .. W / . ,.,_4 PlATE 6. PhotomicrogTaph of a sporangiophore of P. palmivora showing relative sizes of sporangia et different positions in the sympodium. x SOO. University of Ghana http://ugspace.ug.edu.gh 70 FIG. 5 CAMERA LUCIDA DRAWING OF SPORULATING HYPHAL BRANCHES OF MYCELIUM GROWING ON COCOA POD SHOWING POSITION OF SPORANGIA OF DIFFERENT SIZES IN THE SYMPODIUM . University of Ghana http://ugspace.ug.edu.gh 71 G. INDIRECT GERMINATION OF SPoRANGIA OF DIFFERENT SIZES Indirect germination of p. palmivora sporangia in distilled water has been found to be very good at the optimum temperature of 220 C. Clerk (1972) obtained 25 per cent germination in 30 minutes. Percentage germination rose to 70 per cent in one hour and in two hours germination was virtually complete with more than 90 per cent germination. The ability of the sporangia of different sizes to form zoospores was investigated by examining both the rate of germination and the maximuo percentage germina- tion achieved. The necessary observations were made on sporan- gial suspensionsin Petri dishes incubated in light in an air- conditioned room at 22oC. Continuous observations of the germ!- nating sporangia could therefore be made at 15 minutes intervals without altering the tem?erature of incubation. The extent of germination in distilled water after two hours shown in Table 19 at least for sporangia of the two larger size-classes agrees with that reported by Clerk (1972). Within this interval (two hours) 89.7 end 96.0 per cent of the sporen- gia of the med~. size and large-size classes formed zoospores, The maximum percentaee germination was, however, achieved more quickly by sporangia of the medium size-class. Half of the germlnable sporangia in this size class produced zoospores in approximately 30 minutes whilst the same level of germination occurred aftSl:.60 - 75 minutes in sporangia of the large size University of Ghana http://ugspace.ug.edu.gh n Mature sporangia of the small size-class were found to germinate more slowly and to achieve a lesser level, 72.1 per cent, of maximum germination. University of Ghana http://ugspace.ug.edu.gh 73 TABLE 19 Indirect geruination of P. palmivora sporangia of the three size· classes incubated in distilled water at 22°C for 2 hours. *percentage of sporangia forming zoospores Time of Incubation ----- in in Minutes Small size- Medium size- Large size· class sporan· class sporan- class spo· gia giB rangis I ( <24.5 urn) (24.5 -48,S um) (> 48.5 llm) I 15 I 7.0 14.5 9,5 30 21.7 39.1 26.7 45 34.8 62.3 34.1 60 36.1 64.2 38.2 75 46.6 68.9 57.5 90 64.5 84.1 69.8 lOS 63.8 89.1 75.0 120 72.1 89.7 96.0 *Percentage based on a t leas t: (a) 94 sporangia in the Small Size-class (b) 205 sporangia in the Medium size-class (c) 70 sporangia in the Large size-class. University of Ghana http://ugspace.ug.edu.gh 74 H. PRODUCTIVE CAPACITY OF SPORANGIA OF DIFFEREN'r SIZES OuTing observations of the suspensions of the indiTectly germinating spoTangia, diffeTences in the numbeT of zoospoTes emerging from sporangia of different sizes were easily recogni- sed. There is evidence that sporangia of species of other Phytophthoras produce equally varied number of zoospores, Some examples with strikingly wide variations in the number of zoospores formed in a sporangium aTe Phytophthora cinnamoni Rands Bnd Phytophthora megasperma Drechsler which produce from 8 to 40 and 1 to 45 zoospores, respectively, from one sporangium (Waterhouse, 1956). There seems to have been no effort made to relate the number of zoospores formed by the Phytophthoras to the size of the sporangium. A fresh set of sporangial suspension prepared with sterile distilled water was incubated at 22°C fOT 45 minutes. Further development was then stopped by adding a drop of O.lM formalin to each suspension. M!lny sporangia at this stage had produced zoospores which were just about to escape (see plate 7). The zoospores in each sporangium were counted to find out the number of spores contained in sporangia of the different size classes. In the same preparations, activities were also brought to a halt in sporangia in which some of the zoospores had already escaped . (see Plate 8). The remaining encysted zoos~ores in the half-spent sporangium with clearly discernible outlines were used to determine the size of zoospores formed by sporangia of es. The photomicrograph Shows that the University of Ghana http://ugspace.ug.edu.gh 75 formglin used to induce encystment did not cause any noticeable morphological changes. The mean values in Table 20 are averages of zoospores in 40 sporangia and diameters of 100 measured encysted zoospores for each size class. Both the size and number of zoospores varied with sporangial s :l.ze. The number produced by large and small spotangia understand- ably differed. A large-sized sporangium produced on the average 19 zoospores whilst a small-sized sporangium formed only nine zoospores on the average. The number of zoospores formed by a medium-sized sporangium was intermediate between these two extremeE The data of Table 20 was tested statistically using student's t. ~e values obtained were found to be significantly different from e~ch other at 1% level. Sporangia of the Large size-class formed zoospores with a mean diameter of 10.3 pm, which was found to be significantly larger than diameter of zoospores of sporangia of the two other size-classes. The sizes of the zoospores of these two size- classes were almost identical and not significantly different from each other. Each was, however, significantly different at 5% level from the value for the tare size-class. The size of the encysted zoospores of P. palmivora, according to the results obtained here, is similar to that of several other specie.. For example, 7 -10 pm in Phytophthora ~i McRae. and 8 - 10 11m in Phytophthora paeoniae Cooper and Porter, Phytophthora parasitica var. ~ Godfrey and Phytophthora quininea Crandall (waterhouse, University of Ghana http://ugspace.ug.edu.gh 76 TABLE 20 Number of zoospores and size of encysted zoospores produced by P. palmivora sporangia of the three size-classes incubated in distilled water at 22°C. Sporangia 1 Range of lMean No. Mean size-class No. of of zoo- Diameter (based on zoospores spores of encysted sporangial in a in a .zoospores length) sporangium sporangium (lJm) ---- Small 6 - 12 9 9.0 t 0.1 « 24. 5 ~ m) Medium 11 - 18 15 8.9 = 0.1 (24.5 - 48.5 ~m) Large 17 - 25 19 10.3 ± 0.1 (> 48.5 llm) 1 To the nearest whole number. University of Ghana http://ugspace.ug.edu.gh 77 PLATE 1. Photomicrograph showing sporangium of P. palmivora with fully formed and identi- fiable zoospores used in assessing the number of zoospores in the spo~angium. x 100. University of Ghana http://ugspace.ug.edu.gh 78 ...-. PLATE 8. Photomicrograph showing half-spent sporangium of P. palmivora used in estimating the diameter of encysted zoospores of sporangia of known sizes. x 600 .. University of Ghana7 9h ttp://ugspace.ug.edu.gh I. DIRECT GERMINATION OF SPORANGIA OF DIFFERENT SIZES IN DISTILLED WATER Direct germination of sporangia of several !bytophthora species has been shown to be dependent on various environmental factors. Temperature, nutrients, oxygen, pH and growth substan- ces have III been shown to be involved (Aragaki, Mobley and Hine, 1967; Clerk, 1972; Crosier, 1934; Harvey, 1954; Katsura, 1961; Melhus, 1915; Uppal, 1926). One generalisation that arises from these observations is that temperature 1s one of the most important factors determining the mode of germination. Higher temperatures generally encouraged direct germination. Clerk (1972) reported that the s?orangia of P. pa1mivora in distilled water would produce germ tubes in addition to zoospores, at lOoC and at temperatures slightly above this level. Sporangia of ~ palmivora were incubated in distilled water and at 30°C for 24 hours to find out whether the differences in indirect germina- tion observed in Chapter G would occur during germination by means of germ-tubes. The results in Table 21 show that sporangia of the three size-classes germinate almost to the saae extent. Only few sporangia appear to have the capacity to produce germ tubes in distilled water at this temperature. Clerk (1972) also found that only 4.6 per cent of the sporangia germinated by means of germ tubes at lOoe when they were incubated in distilled water and 9.9 per cent did so at 34°c. ,.-.:c.~ sporangia produced not more than four University of Ghana http://ugspace.ug.edu.gh 80 germ tubes which commonly emerged close to the pa?illa. There were apparently differences among the sporangia of the three size-classes in their capacity to form germ tubes. Careful assessment made showed that whereas the majority of the germina- ted sporangia of the small size-class, 91.3 per cent, produced only one germ tube, more than 20 per cent of the germinated sporangia in the two other size-classes had more than one germ tube. The full data have been provided in Table 22. University of Ghana http://ugspace.ug.edu.gh 81 TABLE 21 Germination in distilled wat~r by means of germ tubes by P. palmivora sporangia of the three size-classes incubated at 300 C for 24 hours. S,orangial size- Total No. class based on of sporan- Percentage __ ~a1!B.ia 1 lensth S1a observed G.!!.mination Small 387 7.2 ( < 24.5 llm) Medillm 415 7.0 (24.5 - 48.5 llm) Large 370 7.6 (> 48.5 lID) University of Ghana http://ugspace.ug.edu.gh 82 TABLE 22 Production of germ tdbes by P. palm1vora sporangia of the three size-classes incubated in distilled water at 300d for 24 hours. -------- Sporangisl 'rotal Percentage of germinated aize-class No. of sporangia bearing indicated (based on germinated number of germ tubes sporangia1 sporangia length) 1 2 I 3 4 Smell 219 91.3 8.' I 0.0 I 0.0 «24.S lJm) I I I I Medium 446 10.4 24.2 4.S 0.9 (24.S .. 48.5 tim) I I Large 2'8 68.1 26.1 4.6 1.2 ( >48.5 tJm) University of Ghana http://ugspace.ug.edu.gh 83 J. AMJUN'r OF HYPHAL GROW S UPfORTED BY ENDOGENOUS §J!!!!TRATml .Q! sroBANGtA OF PIFFERENt SlZES Spores of fungi which germinate readily in distilled water have obviously sufficient nutrient reserves ab ~ to support the germination processes. There are ecological advantages if the endogenous nutrients occur in sufficient quantities to support also growth of the germ tubes. Spores of that sort possess a greater potential for survival than those that need exogenous nutrients for growth of the germ tubes inmediately they emerge. It was considered necessary to examine growth of the germ tubes of P. palmivora preduced in distilled water to find out whether greater sporangia size was accompanied by greater growth of the germ tubes because of the presence of larger stores of reserve nutrients. A sporangial suspension of p. palmivora of very low density. 1,000 sporangia per suspending medium, was incubated at 30oe. This density was deliberately selected to improve the chances of measuring as much as possible germ tubes of the same sporangia throughout the inve.tigation.~om ~ earlier report of the prece- ding chapter. sporangia which would produce germ tubes would be approximately only seven per cent of the population. 'l1le germ tubes of 20 sporangia of each size-class were measured at 2 hour intervals. In order to obtain a complete cycle. two SPt-1I "f 9"apension were prepared, the first set was University of Ghana http://ugspace.ug.edu.gh incubated at B.oo atm. on the day the experiment started, and the second suspension was prepared and incubated at 8.00 p.m. the same day. Readings of the second set taken from 8.00 4.m. to 8.00 p.m. the following day oonstituted growth from 12 to 24 hours. The two sets of readings nicely fitted together to provide growth curves of 1ig.6. There was seemingly no growth after 24 hours and the readings taken have therefore been omitted from the results in Table 23. Where a sporangium produced more than one germ tube, the record for the sporangium was the sum of all the germ tubes. The measurements in Table 23 show that sporangia of the Medium aad Large Size-classes possessed elosely similar quantities of nutrient reserves since they supported hyphal growth to the same extent. The germ tubes of the sporangia of the Small size-class were shorter at each stage, indicating that the sporangia contained lesser levels of nutrient reserves. University of Ghana http://ugspace.ug.edu.gh 85 TABLE 2~ Growth rate of germ tubes of p. palmivora sporangia of the three 0 siZe-classes in distilled water at 30 C. -------1 EXPERIMENTAL SET I Mean Length of germ tubes (um) produced by sporangia Tirrle of Incuba- I --- tion in Small MediB'" Large Hours class-size class-size size-class I sporangia sporangia sporangia ( < 24.5 \lm) (24.5 -48.5 lJm) (:> 48.5 IJ til) 2 70.1 98.7 97.8 4 180.2 231. 7 276.0 6 198.8 245.3 274.0 8 267.4 320.3 ~63.1 10 451.9 517.7 507.7 12 514.0 707.9 687.7 University of Ghana http://ugspace.ug.edu.gh 86 TABLE 23 (cont'd.) Growth rate of germ tubes of P. pa1mivor~ sporangia of the three o size-classes in distilled water at 30 C. EXPERIMENTAL SET II Mean Length of germ tubes ( lJm) produced by sporangia Time of Incuba- tion in Small Medium Large Hours class-size class-size size-class sporangia sporangia sporangia ( < 24.5 llm) (24.5 - 48.5lJm) (> 48.5 lJ m) 12 603.5 783.0 670.0 14 672.1 855.1 973.0 16 989.6 1124.0 1189.0 18 1068.2 1225.4 1234.0 20 1051.0 1389.8 1268.0 22 1287.0 1464.4 1430.0 24 1278.0 1449 •.2 1408.0 University of Ghana http://ugspace.ug.edu.gh 87 1500 o 1000 500 .. ·x. . ·SMALL SIZE-CLASS -o-MEDIUM SIZE-CLASS ___ LARGE SIZE-CLASS o 4 8 12 16 20 24 28 INCUBATION TIME IN HOURS FIG . 6. GROWTH OF GERM TUBES FORMED BY GERMINATING P.PALMIVORA SPORANGIA OF THE THREE SIZE -CLASSES IN DISTILLED WATER AT 30DC . University of Ghana http://ugspace.ug.edu.gh 88 K. FORMI\TION OF S~ONDARY SPORANGIA BY SPORANGIA OF DIFFERENT SIZES In a number of fungal species, growth of the germ tube is limited and a mycelium is not produced. The germ tube directly gives rise to a secondary spore. This phenomenon is unusually common in certain groups of fungi, notably in the order Entomo- phthorales (Drechsler, 1947, 1953. 1954. 1955a. 1955b. 1960; Dring, 1958; Page and Humber, 1973; etc.) and in the Genus Phytophthora (Hotson and Hartage, 1923; waterhouse. 1956; etc.) The secondary sporangium in the thytophthoras is useful as a dispersal and infection unit rather than a survival body because of its fragility. The ability of a sporangium to pro- duce a secondary sporangium must be related to the vigour of the primary sporangium. The previous experiment has shown that sporangia of P. pal~ora of the small size-class did not support the same amount of hyphal growth. It is possible that sporangia of this category would also possess lower potential for secondary sporangium development. This supposition was examined in a subsequent experiment. P. palmivora sporangia suspended in distilled water were incubated at 300 e and the proportion of germinated sporangia of each size class which formed secondary sporangia was determined. P. palmivora formed secondary sporangia in similar pattern as other species of Phytophthora which have been _ •. L- of not more than 160 - 200 11m long stopped University of Ghana http://ugspace.ug.edu.gh 89 growth and develo?ed at its apex a secondary sporangium of exaetly the some shape as the primary sporangium (see plate 9). The mediom and large size-classes contained a greater percentage, 54.0 and 53.4 per cent, respectively, of germinated sporangia with secondary sporangia than the small size-class, 43.3 per cent (Table 24). Consistently, the secondary sporangium never developed to the same size as the primary sporangium, and at maturity was significantly smaller. The sizes of the secondary sporangia produced in each size-class are shown in Table 25. A significant observation was made. The extent of reduction in size of the secondary sporangia differed with the size-class. This is well b illustrated by the mean secondary sporagium length I mean primary A sporangium length ratios, The difference in size between the secondary and ?rimary sporangia diminished with decrease in size of the latter, University of Ghana http://ugspace.ug.edu.gh 90 TABLE 24 Formation of secondary sporangia in distil1ed wotef by directly germidating p, pnim!vora sporangia of the three ~i~e.cia~Be9 incubated at 300C for 24 houre, Sporangia1 she- Total No. of Percentage of class based on germinated germinated sporangial length sporangia sporangla with observed secondary sporangia Small 120 ( <24.5 Um) Medinm 126 54.0 (24.5 - 48.5 \.1m) Large 120 53.4 (> 48.5 um) University of Ghana http://ugspace.ug.edu.gh 91 Size (Length) of mature secondary sporangia formed by .~~ sporangia of the three size-classes incubated in distilled water at 30°C. ---------- Sl£e-class No. of Mzan Mean secondary of Secondary Length Sporangium Length/ Primary Sporangia of Mean prlr.Jary 1 Sporangia Measured Secondary s?Orangium Length Sporangia ratio in tlm ----- Small 34 21.0:! 1.4 0.99 « 24.!i lot t!l long) (21.0 /21,6 \.1m) Mediam 65 25.3! 1.3 0,69 (24.5 - 48. 5 tJIll (25.3 /36,3 lJD.) long) Large 20 32.4 ± 1.4 0,63 (> 48. S.\Im long) (32.4 /H.B lim) 1 Mean of 50 Primary Sporangia. University of Ghana http://ugspace.ug.edu.gh 92 fo ..Q o M (' . PlATE 9. Photomicrograph of sporangia of p. palmivora of different size-classes producing secondary sporangia in distilled water at 3oPe. x 90. Note differences in size of secondary sporangia produced by the Large-sized (L), Medillm-sized (M) and Small-sized (S) primary sporangia. University of Ghan9a3 http://ugspace.ug.edu.gh L. GERMINt\TION OF spoRANGIA Of DIFFERENT SIZES IN COCM POD EXTRACT In studies concerning physiology of fungal pathogens, the influence of the host is of immense importance. In distilled water, sporangia of P. palmivora of different sizes varied in some aspects during germination. For example, the rate Bnd extent of zoospore formation were greater in sporangia of the MediUM and Large siZe-classes than those of the Small size-class. Of much relevance to infection of cocoa by P. palmivora would be compounds found on the CQcoa pod and roots of the cocoa tree, organs which constitute the principal sites of initial attack by the fungus. It was important to find out whether the sporangia of different sizes in the infection court will respond differently to each .compounds. The effect of extract of the cocoa pod husk, which will affect sporangia infecting the pod through wounds in the field, on the germination of the sporangia was firs t examined. Green cocoa pods Were used to prepare husk extracts at two concentrations, 0.5g husk tissue per 20 ml distilled water and 1.08 husk tissue per 20 ml distilled water and used BS germina- tion media. Sporangial su.spensions prepared with the extracts were incubated Bt lOoC. The incubation temperature together with the component nutrients of the extract would encourage direct germination. In order to make accurate record of germi- nation, especially with respect to sporangia of the Small and normally took much time to locate, University of Ghana http://ugspace.ug.edu.gh sever~l Petri dishes were set up and samples were withdrawn at hourly intervals for percentage germination assessment of sporangia of each size-class. Further development was stopped immediately on removal by adding a drop of O.lM formaldehyde to the sporangial suopension. Each value in the table of results was based on 200 observed oporangia (Table 26). The results in Table 26 show that the extract at a concen- tration of 1.Og husk tissue per 20 ml distilled water was the better medium for germination of the sporangia of all sizes. Hundred per cent germination was obtained in all the suspensions after four hou~s. The rates of germination of sporangia of the different cize-c1asses in this medium were quite similar. At the less favourable concentration of 0.5g husk tissue per 20 m1 distilled water. germination of sporangia of the Small oize-class was lower than those of the other two size-classes. After three hours, percentage germination in the Small, MediUM and Large size-classes was 44.3, 55.8 and 57.9 per cent, respec- tively, and at the end of the experiment there was 88.7 per cent germin~tion in the Small size-class in contrast With 100 per cent germination in the other size-classes. University of Ghana http://ugspace.ug.edu.gh 95 TABLE 26 Germination by m2an3 of germ tubes of P. palmivora sporangia of the 0 three size -·classes incubated in cocoa pod extract at 30 e. I Sporangia 1 I Concen- Percentage germination after size-class I tration indicated hours of incuba -(based on of tion sporangia 1 I Extract pH length) I (g ./20ml H2O) ~-t 2 3 4 I I 0.5 5.6 7.7 I 20.0 44.3 88.9 Small 1.0 5.6 11 . 5 39.0 81.8 100 0.5 5.6 10.1 49.0 55.8 100 Medium 1.0 I 5.6 18.3 52.9 85.0 100 ---- 0.5 5.6 20.8 30.8 I 57.91 100 Large I ! 1.0 5.6 14.3 50.0 90.9 L1 00 - University of Ghana http://ugspace.ug.edu.gh 96 M. EFFECT OF COCOA. ROOT EXUDATE ON GERMINATION OF SPORANGIA OF DIFFERENT SIZES The effect of exudate of cocoa root was next examined. The exudate& of plant roots are mixtures of several compounds, principally, carbohydrates, amino acids, enzyme8, organic acids and growth factors. The effect of the exudate on fungi in the rhizosphere is not uniform. It could be either stimulatory or inhibitory, For example, mycelial growth in Pellicularia filamentosa (Pat.) Rogers was stimulated by root exudates of radish, Raphanus ~ Linn. and lettuce, Lactuca ~ Linn. (Kerr, 1956), and, inhibited in several fungi. Byssochla- !l! ~ Westling, Colletotrichum atramentarium (Berk. and Br.) Taubenh., pythium rnamillatum Meurs and Spongospors subterranea (Wallr.) Lagerh, by root exudates of oats. ~ ~ Linn. (Schone beck. 1958), tomato, Lycopersicum esculentum Mill. (Ebben and Williams, 1956). turnip, Brassies esmpestris Linn. (Barton. 1957) and potato. ~ tuberosum Linn. CWhite. 1954), respectively. Conidia of Fusarium oxysporum f. ~ (Smith) Snyd. and Hans. were encouraged to germinate by root exu- date of!banane (var. Gros Michel), Musa sapkfttum (Buxton. 1962) Banana (var. Lacatan) and peas, ~ ~ Linn. root exudates on the other hand, inhibited gerwination of Fusarium oxysporum f. ~ and Fusarium oxysporUl7i f. pisl (Unf.) Snyd. and Hans, conidia (Buxton. 1957)~ These differences are due to differences in the nature of the exudates. ~here is stimulation when " -""",,""'Y"'I&JU"'''',:'Otiter proportion of the components. Effects University of Ghana http://ugspace.ug.edu.gh 97 of the nutrients could, however, be over-shadowed by toxic compounds. such as organic acids 1n the event of inhibition. Organic acids in addition to their direct effect on the fungi have secondary effects, such as modification of pH of the rhizosphere and chelation of metals. The effect of root exudate of cocoa on germination of sporangia of ~~ generally, and in particular on germination of sporangia of different sizes, was examined in the following experiment. Seedlings of cocoa supported by cotton-wool plugs with their roots in sterile distilled water and grown at 2SoC provided root exudates for the germination experiments (Plate 10). The te.ta of seeds of freshly harvested ripe cocoa pods was carefully removed. The seeds were then surface sterilised by immersing them for one minute in 1.0 per cent Mercuric chloride solution and rinsing in several changes of sterile distilled water. Each seed was then placed upright in a small depression in cotton-wool plug of a 50 ml flask containing 30 ml sterile distilled water and allowed to germinate. The flasks were then kept in a perspex chamber during both germination of the seeds and growth of the seedlings. The seedlings were given normal day/night light regime. The flasbwere covered with aluminium foil to keep the roots in continuous darkness. The TBdicle reached the water within 5 days and the exudate" used in the test was produced in approximately 15 days. University of Ghana http://ugspace.ug.edu.gh Sporangia1 suspensions were prepared with the contents ot the flasks, both directly and at a lower concentration of 1t1 dilution and incubated at 300 e to encourage direct germination. Germination of the sporangia in the exudates at the concen- trations used was not very high as indicated in Table 27. Germi- nation of sporangia of the Medium and Large size-classes after four hours ranged between 30.0 and 34.0 per cent which was anyway greater than germination of sporangia of the Small lice- class, which at the better concentration, undiluted medium, was 21.7 per cent. Readings were not extended beyond four hours because of the extenGive growth of the germ tubes. Control suspensions prepared with distilled WBter gave direct per cent germination of 6.8, 7.0 and 6.9, respeetlvely. for sporangia of the Small, Medium and Large size-classes after four hours incubation. University of Ghana http://ugspace.ug.edu.gh 99 TABLE 27 Germination by means of germ tubes of ~l~sporangia of the three size-classes incubated in root exudates of 20-day old cocoS seedlings at 30oc. Spceangia1 Concen- Percentage germination size-class tration after indicated hours (based on of pH of incubation sporangia1 Exudate length) 1 I 2 3 4 I 1:1 Dilution 8.2 I 0.0 I 6.4 15.1 17.8 Small « 24.5 llm) Undiluted 8.5 0.0 9.5 20.5 21.7 - ~ 1:1 Dilution 8.2 0.0 5.9 16.8 31.8 Medium (24.5 -48.5 \lm) Undiluted 8.5 0.0 9.7 19.4 30.0 - -. ~-- _1.; 1 Dilution 8.2 0.0 5.8 18.5 32.7 Large (> 48.5 llm) Undiluted 8.5 0.0 11.5 25.8 34.0 University of Ghana http://ugspace.ug.edu.gh 100 PlATE 10. Photograph of 10-clay old cocoa seedlings growing in sterile distilled water for the production of root exudates. x \ University of Ghana http://ugspace.ug.edu.gh 101 N. AN.:\LYSIS OF Roar riT.TDATE OJ! COCoA The cocoa pod extract was a far superior germination medium to the root exudate. There could be several reasons for this. The extract contained nutrients in greater quanti- ties, contained types of nutrients which were more stimulatory, or contained lesser quantities or no inhibitory substances. The e>:tract has been an:llysed by Bimpong (1969) by paper chromatography. She reported that "results of several tests made for the pr.esence of carbohydrates in the extract were negative". Four. amino acids, asparagine, glutamic acid, glycine and tyrozine at the respective concentrations of 2000, 3000, 400 - 500 and 200 - 300 p. p.m. were identified. For purposes of comparison, the exudate prepared for the experiment of the previous chapter was analysed for the presence of amino acids and carbohydrates. The root exudate ~;as chromatographically analysed. Developed chromatograms were tested for carbohydrates (Plate 11). From a series of chromatographic tests (12 tests) the constituent carbohydrates in the exudate were identified and their respective quantities estimated. The results are presented in Table 28. Quantitative estimates were based on visual comparisons of the spots (of identified carbohydrates) with spots produced by known quantities of corresponding markers. Five ca~bohydrates, galactose, glucosamine, glucose, - -entifieo. University of Ghana http://ugspace.ug.edu.gh 102 'MBLE 28 Component carbohydrates of root-exudate of 20 day-old cocoa seedlings identified by paper chromatography. (Cocoa seedlings grown in continuous light of 250 lux) Carbohydrate Approximate quantity in Ilg/ml. G-:llllctose 1- 30 -35 Gluco3amine 30 - 3S Glucose I .so ..- S5 Lactose IS - 20 Ribose 15 - 20 University of Ghana http://ugspace.ug.edu.gh 103 PLATE 11. Photograph of developed chromatogram showing separated constituent carbohydrates of root exudate of 20-day old cocoa seedlings. The marker on the right is Glucose. University of Ghana http://ugspace.ug.edu.gh 104 The root exudate was analysec mechanically with an Amino Acid An3lyser Model JLC-6AH in the laboratories of the Department of Chemical Pathology. University of Gh3n3 Medical School. The Amino Acid Analyser identified the amino ccids and determined their respective quantities. Fig. 7 is a xerox copy of the data sheet of the Ar.alyser. Eleven amino acids were identified of which three, asparagine, glycine and tyrosine also occurred in the extract of the cocoa pod husk. The eleven amino acids and their concentrationo in the exudate have been tabulated in Table 29. Before the root exudate was used for both analyses, contents of several flasks with growing cocoa seedlings were pooled and agitated very thoroughly with mechanical shaker (Griffin and George Ltd., England). An amount of 500 ml was then concentrated to 5 ml using a Simple distillation apparatus. The exudate was heated in a water bath maintained at a temperature of 40°C. The outlet of the distillation apparatus was connected to a suction pump which reduced the internal pressure to allow bolling at 40°C. University of Ghana http://ugspace.ug.edu.gh lOS TABLE 29 Constituent Qmin~ acids of Root.exudate of 20 day~old cocoa aeedlings analysed mechanically with l~in~ Acid AnalysEr HOdel Jl£-6AH. (Cocoa seedlings grown in continuous light of 250 lux) Peak No. Amino acid Concentra don (Ilmole/ml. ) 1 Asparagine Tr'8ce 2 Threonine 0.002 3 Serine 0.012 4 Glycine 0.004 5 Alanine 0.005 6 Valine Trace 7 Isoleucine Trace 8 Leucine Trace Ammonia 0.002 10 Histidine 0.004 11 Tyrosine 0.015 University of Ghana http://ugspace.ug.edu.gh 107 O. EFFECT OF NITRogENOUS COMPOUNDS ON GERMINATION OF SPORANG:-A 01 D~E3.r;~~ SIZES Despite the presence of both amino acids end carbohydrates in the cocoa root exudate, the extr~ct of cocoa pod husk with either no carbohydrates or more probably with carbohydrates in concentrations too low to be detected by peper chromatography and with reportedly fewer amino ncids l::lS a better medium for direct germination of the sporangia. Compounds in the husk extract other than amino acids and carbohydrates might have played an im~ortant role in the stimulation of germination of the sporangia. It was presumed that any stimulation by the amino acids and carbohydrates individually may be too low to bring out clearly any differences in the response of sporangia of different sizes to nutrients. Response of the sporangia was therefore investigated using complex compounds in order to promote subs tan- tial germination. The nitrogenous compounds, Casein hydrolysate, Peptone and Yeast-extract were selected and used to find the effect of nutrients on direct germination of the sporangia. Besides the large number of amino acids contained in each com- pound, the proteins have been chosen because they are composed of substances common to both the cocoa pod husk extract and cocoa root exudate. Each compound was tested at two concentrations, 0.5 and 1.0 per cent (w/v). Sporangi.R) !luspenc~ons prepared with the various University of Ghana http://ugspace.ug.edu.gh 108 tion. There were 16 Petri dishes of each suspension and four were withdrawn at hourly intervals for determination of percen- tage germination of sporangia of each size-class. Each percen- tage germination in Tables 30 to 32 was based on 200 observed sporangia. A concentration of 1.0 per cent was found to be the better medium for sporangial germination. The response of the sporan- gia, based both on the rate of germination and on the final percentage germination after four hours incubation, generally varied with sporangial size. The large sporangia always bene- fitted most and the small sporangia least from stimulation. A 8ignificant feature was the different effects of the three compounds on germination of sporangia of the different slze-class~. variation in response of the sporangia of the three size-classes to peptone was slight (Table 31). It was, however, very Wide in casein hydrolysate and yeast-extract solutions, particularly, in the less favourable medium of 0.5 per cent concentration (Tables 30 and 32). For example, in the casein hydrolysate , solutions , the germination rates of 31.8, 48.5 and 75.8 per cent were recorded after four hours for sporangia of the Small, Medium and Large size-classes (Table 30). The results in the three tables appear together in Fig.8 for comparative purposes. University of Ghana http://ugspace.ug.edu.gh 109 TABLE 30 Germination by means of germ tubes of P. pa1mlvora sporangia of the o three size-classes incubated in Casein hydrolysate solution at 30 e. Sporangia1 Concen- I Percentage germination size-class tration I after indicated hours (based on of pH of incubation sporangia 1 Medium I length) % (w/v) I I 1 2 3 4 - - 0.5 6.0 0.0 0.0 9.4 31.8 SlDSll ~- ( < 24.5 ~m) 1.0 6.1 0.0 0.0 28.6 56.8 I 0.5 6.0 0.0 0.0 9.2 48.5 Medium (24.5 -48.5 \.lID) 1.0 6.1 0.0 0.0 31.3 55.1 0.5 6.0 0.0 0.0 6.1 75.8 Large (>48.S~m) --r-- 1.0 6.1 0.0 I 0.0 26.3 77.9 --- University of Ghana http://ugspace.ug.edu.gh 110 TABLE 31 Germination by means of germ tubes of p. palmivora sporangiA of the three size-classes incubated in Peptone solution at 3QOC. Sporangial Concen- Percentage germination size-clsss tration after indicated hours of (based on of pH incubation sporangia1 Medium length) '1. (w/v) 1 2 3 4 - 0.5 5.8 0.0 2.9 11.4 45.2 Small « 24.5 lim) 1.0 5.8 0.0 2.2 17.4 49.2 0.5 5.8 0.0 4.6 13.3 52.1 Med11!nI (24.5 - 48.5 1I m) 1.0 5.8 0.0 7.5 21.0 54.0 ---- 0.5 5.8 0.0 1.7 12.9 54.5 Large (>48.511m) 1.0 5.8 0.0 6.2 19.6 55.0 University of Ghana http://ugspace.ug.edu.gh 111 TABLE 32 Germination by meana of germ tubes of p. palmi. ... J ::'t; sporangia of the three size-classes incubated in Ye8st-extract sOLution at 30°C. Sporangial Concen- Pc=ccntage germination size-class tratlon after indicated hours (based on of pH of ir.,:ubation sporangial Medium length) '? (w/v) 1 I 2 3 4 0.5 7.2 0.0 2.9 4.1 27.7 Small « 24.5 l!m) - - 1.0 7.7 0.0 7.8 33.3 79.2 0.5 7.2 0.0 3.9 5.8 55.6 Medium (24.S - 48.5 IJ m) 1.0 7.7 0.0 7.9 42.0 85.9 0.5 7.2 0.0 4.5 6.3 59.2 Large (> 48.5 l!m) 1.0 7.7 0.0 8.5 51.4 89.2 University of Ghana http://ugspace.ug.edu.gh 112 z 100 0'5 PER CENT 1'0 PERCENT o t-( 80 _ CASEIN HYDROLYSATE ~ :~~ ~ ILl ",..,rorri-I+++I+I ~:'.'.'.'. '" 20~:::::::: :.:.1:.::.:.;.;.;.;.; ~ Or::~::::~:::~:~~--~-4~~~~L-~ z 100 o ~z aOr- PEPTONE 6010- ~ - ct 40~;······· ........ . ILl '" 2 0 J;;:;:;:~: ~::~.:~::'.~~:~~.~~.:.~.~'.~. ~ 0~:;~:~~~~~:~~~--~-4~~~~L-~ YEAST EXTRACT FIG. a. GERMINATION OF P. PALMIVORA SPORANGIA OF THE THREE SIZE-CLASSES BY MEANS OF GERM TUBES IN 0-5 AND I· 0 PER CENT SOLUTIONS OF NITROGENOUS COMPOUNDS AT 30o C. IN 4 HOURS. SIZE. -CLASSES : ~ SMALL; EEIJ MEDIDM ; CJ LARGE. University of Ghana http://ugspace.ug.edu.gh lil f. DF.VELOPHENT or GERM TUBES BY S PORAOOr;. OF DIFFERENT SIZES IN NUTRIENT MEDIA The infection capacity of spo~angi8 of the different slze- classes depends on two important factors. '!be percentage of the population that ca" germinate and, in indirectly germinating sporangia, the number of zoospores produced by a sporangium. In the case of directly germinating sporangia. the second factor is the number of germ tubes a germinating sporangium could produce. The potential for infection is naturally greater when several germ tubes emerge; the sporangium thus becomes a 'many-pronged' weapon. During the previous investigation, the first condition - percentage germination was assessed. The importance of a nutrient that encourages high germination rate becomes even greater if it would also stimulate the production of large number of germ tubes. The development of germ tubes of sporan- gia of different sizes under the influence of nutrients was therefore investigated. only three media were chosen for this experiment. Yeast- extract at a concentration of 1.0 per cent, in which sporangia of different sizes germinated very well and 0.5 and 1.0 per cent Casein hydrolysate in which considerable variation in germination rate was observed (see Fig.8). Sporangia from inoculated cocoa pods incubated in these solutions at 30°C were examined after six hours aod the number University of Ghana http://ugspace.ug.edu.gh 114 of germ tubes of each sporangium in the microscope field was noted. Several random observations were made, giving finally a total of not less than 200 germinated sporangia for each size- class. The germinated sporangia of each size-class were separated into three groups or categories according to the number of germ tubes; Category I, 1-3 germ tubes; Category 2, 4-6 germ tubes, and category 3, more than 6 germ tubes. The values obtained for each size-class on each solution are shown in the 3-dimensional graphs in Fig.9. Generally different media had different effects on the development of germ tubes by sporangia of a particular size- class. The number of germ tubes increased with increasing size in sporangia, in solutions of 1.0 per cent Yeast-extract and 0.5 per cent Casein hydrolysate. In the former the respective per- centages of germinated sporangia in the S~ll-sized, Medium- sized and Large-sized classQS with four or more germ tubes were 19, 25, and 73 per cent, and in the casein hydrolysate solution, So, 70, and 67 per cent. In contrast, the number of germ tubes was greater in the Small-sized sporangia germinating in 1.0 per cent Casein hydrolysate solution. Approximately 65 per cent of the Small-sized sporangia had four or more germ tubes per sporan- gium whilst the values for the Mediu~-sized and Large-sized sporangia were 58 and 39 per cent, respectively. The percentage germination of sporangia of the different aize-clAAAPD nhDO_'~~ ., this experiment was almost identical in _----:: :.:.~s of the previous chapter. University of Ghana http://ugspace.ug.edu.gh 0'5% CASEIN HYDROLYSATE « 6 ~ 1'0 % CAS£IN a: o HYDROLYSATE Q. III o 0- « Gz « (( o 1'0 % YEAST-EXTRACT Q. III o 0- o >6 ~~ 4-6 -<,.-V~ ~~ L 1-3 f(~f(; ~O.o FIG.9 . NUMBER OF GERM TUBES FORMED BY P,PALMIVORA SPORANGIA OF DIFFERENT SIZES (S NlUM HYDRO- GEN PHOSPHATE [(~)2HP04J SOLUTIONS The sporangia of P. paLnivora used extraneous nutrients for complete germination by means of germ tubes. Sporangia of the small siae-class were unable to use nutrients supplied externally to the same extent as those of the other two slze-classes. Three possible reasons could be suggested. Permeability of the mem- branes may be inferior. The endogenous reserve nutrients may. on the other hand, be very law. or. metabolism of the small-sized sporangia may be less efficient. By employing inhibitory compounds it would be possible to find which of these suggestions is operating here. It may be deduced in the event of a more severe effect on sporangia of the small size-class that there was adequate ingress of the chemicals into the sporangia snd the inhibitory compound may be acting either by disrupting to a greater extent a les8 efficient metabo- lism or over-shadowing more readily a low concentration of nutri- ent reserves. This supposition was investigated using two mineral salts, CaC12 and (NH4)2HP04' which preliminary studies have shown to suppress zoospores formation to varying degrees over concen- trations of 1.0 to S.OmM. It was not possible to use any compound containing copper which is an important component of many fungi- Cides, because the sporangia were found to be extremely sensitive . ~late at even a concentration of O.OlmM University of Ghana http://ugspace.ug.edu.gh 117 was totally inhibitory. Sporangia I suspension in cae12 and (NH4)2HP04 solution at concentrations shown in Tables 33 and 34 were prepared. Half the nu~ber of Petri dishes (four) for each treatment was incuba- ted at 220 e and the other half at lOoe. for six hours. Zoospores were formed at all concentrations of both caCl2 and (~)2HP04 (Table 33). Germination in distilled water was better than in any of the salt solutions. The depressing effect of both C8C12 ·and (~)2HP04 affected more severely sporangia of the Small Size-class than the others, indicating that the mem- branes were freely permeable to chemicals. An interesting observation was the contrasting effect of the two mineral salts on direct germination When the sporangia were incubated at 300 e (Table 34). Whilst eeC12 was completely inhibitory at all concentrations tested, (NH4)2HP04 was in fact stimulatorY ,supporting direct germination at all concentrations far higher than what occurred in distilled water. The effects of casein hydrolysate peptone and yeast-extract were confirmed here. Sporangia of the Large size-class were stimulated far more than those of the two smaller size-classes. University of Ghana http://ugspace.ug.edu.gh 118 TABLE 33 Effect of CaC12 and (NH )2HP04 solutions on germinatlon of P. palmivora 4 sporangia of the three size-classes by means of zoospores at 220 C in 6 hours. Percentage germination of sporangia in size group Mineral Concen- (sporaogial size based on Salt trotton pH length) (111M) Small Mediam ~rge «24.5 lJm) (24.5 - 48.S11m) (>48.>\lIIl) 2.0 5.3 67.1 82.7 88.4 3.0 5.5 50.0 74.5 68.7 CaCl2 4.0 5.5 50.8 58.6 62.5 5.0 5.5 52.0 61.4 68.3 2.0 7.5 46.0 78.0 81.0 3.0 7.5 47.0 55.0 66.0 (NH4)2HP04 4.0 7.5 17.0 29.0 45.0 5.0 7.5 0.0 0.0 --- 0.0 Distilled Water - 7.0 78.6 88.9 - 92.6 University of Ghana http://ugspace.ug.edu.gh 119 TABLE 34 Effect of C8C12 and (Ha4)2HP04 solutions on germination of p. palmivora 0 sporangia of the three size-classes by means of germ tubes at 30 e in 6 hours. Percentage germination of sporangia in size group (sporangial size based on Hineral Concen- length) Salt tration pH (mH) Small HediQm Large «24.5 \.1m) (24.5 -48.518) (>48.5lJm) 2.0 5.3 0.0 0.0 0.0 3.0 5.5 0.0 0.0 0.0 caC12 4.0 5.5 0.0 0.0 0.0 5.0 5.5 0.0 0.0 0.0 2.0 7.5 34.0 34.8 55.0 3.0 7.5 27.9 28.6 41.4 (NH4)2HP04 4.0 7.S 28.6 29.4 41.7 5.0 7.5 19.4 24.1 44.7 Distilled water - 7.0 7.7 9.0 9.5 University of Ghana http://ugspace.ug.edu.gh 120 R. MITOCHONDRIA. RNI\ AND NUTRIENt' RESERVES IN S PORANGJA OF DIFFERENr SIZES The suggestion in the previoUS chapter that nutrient reserves may occur at different levels in sporangia of different sizes snd therefore account for the differences in germination rate was investigated in a ltmited experiment here. The sporan- gia were stained appropriately to find out the levels of the major nutrient reserves, lipids and proteins. Unlike the majo- rity of fungi, the genera of Oomycetes contain no glycogen, instead, a solUble carbohydrate - glucan - is the .torage carbo- hydrate. The cytoplasm of Pbytophthora palmi,vora is rich in soluble B-(l"3)-glucans of which two different types can be recognised. The glucans extracted from the mycelium are all neutral molecules. In the asexual repreductlve stages (sporan- gia, zoospores and cyats) neutral glucsn is also present but the main component is a family of novel phospboglucans which consti- tute as much as 20 to 2S per cent dry weight. The sporangia contain Phosphoglucan fractions P-I. P-II and P-III and the zoospores P-I, P-II, P-III and P-IV (WOng and Bartnicki-Garcia. 1973). Although cellulose occurs in the cell wall of these fungi, the predominant polymer is a highly insoluble. noncellu- losic, B-(l +3)-glucan with branches at C-6 and any staining for glucan in the sporangium would be obscured by stained glucan in the cell wall. No examination of glucan content was therefore made. Tn t-h ......" ' ... ~ ~~aining was extended to find out the concen- University of Ghana http://ugspace.ug.edu.gh 121 aation of two important cell components, mitochondria, which are so vital to respiration and ~ because of its priocipal role in protein synthesis, both events being indispensable to fungal spore germination processes. The procedures adopted are fully described in the Materials and Methods and the results could be summarised 8S follows: Nutrient Reserves 1. Lipids Lipids, as expected from published ultrastructural micr~ graphs of aporangia of Phytophthora (Chapman and Vujicic, 1965; Clerk. 1974; Elsner, Borton and Bowen. 1970; Hemmes and Hohl. 1969; Williams and Webster, 1970), appeared as stained globules clearly observable especially under oil immersion (Plate 12). The lipid globules were evenly distributed and occurred at the same concentration in sporangi8 of different sizes, Their numbers were too large to permit count of the lipid globules. Since the densities of the lipid globules of sporangia of different sizes were the same. it follows that the larger the sporangial body the greater the number of lipid globules it would contain. 2. Proteins Sporangia stained for proteins were deeply coloured indicating heavy concentration of proteins. The 8taining was ,rotop1ast and no differences in concen- University of Ghana http://ugspace.ug.edu.gh 122 tration of proteins could be detected. Larger sporangia, therefore, with the same concentration of lipids and proteins as smaller sporangia would have greater amount of these nutrients at their disposal for germination and germ tube growth. Hi tocbond ria The sporangium of p. palmivora is known from ultrastructu- ral studies to contain a beavy concentration of mitochondria (Clerk, 1974). This was confirmed by the histochemical tests. plate 13 illustrates, first, the deep staining of the sporangia indicating the high density of mitochondr1s , and, secondly, the identical intensities of the staining in sporangia of different sizes. K;N.A. Protein synthesis mey occur at different rates in sporangia of different Sizes, not because of any differences in density of ~ PQr unit volume of the sporangium since the same intensities were observed on treatment with the appropriate stains, but because of the greater volume available to large sporangia. University of Ghana http://ugspace.ug.edu.gh 123 PlATE 12. A\otomicrograpb of sporangia of p. palmivora of different sizes stained with Sudan III to show lipids in the cytopla8~ x SOD. University of Ghana http://ugspace.ug.edu.gh 124 \,,/..\.r~< .. ! . 4-'" \ I' \ /' /''-- --'~ - A • PlATE 13. ibotomicrograpb of 'pol'4ngia of P. palm1vora of different sizes stained with 0.01 per cent Janus Green to show concentration of mitochon- dria. x 600. Note identical intensity of stain in sporangia of different slse8. University of Ghana http://ugspace.ug.edu.gh 125 s. SUSCEPl'IBnITY OF SPORANGIA OF DIFFERENT SIZES TO' DESICCATION It has been observed that death came earlier to sporangia of the small size-class than those of the other size-class8s when inhibitory levels of mineral salts were applied. Death of sporangia under those conditions would be vastly different from destruction of the spore through desiccation since completely different processes would operate. The behaviour of the sporangia of different sizes in the atmosphere on cocoa farms cannot therefore be inferred from the results of Chapter'P: Sporangia of Phytophthora are reportedly very short-lived in air. The most striking effect of lower humidities on viability of sporangia of 8 Pbytophthora species was reported by Warren and Calhoun (1975). They obllerved that all the sporangia of Pbytophthora infestens (Jotlnt) de Bary exposed to an atmosphere of 95l R.B. did not live for more than 20 minutes and, in fact, only 30 -40 per cent were viable after exposure to this relative humidlty for five minutes. At 90% R.B. death of the entire population occurred in exactly six minutes. To examine the effect of this immensely important ecological factor - desiccation - in the life of the sporangia of P. palmivora of different slaes, the viability of the sporan- gia was investigated USing relative humidities comparable with those recorded in cocoa farms and reproduced below in Table 35. University of Ghana http://ugspace.ug.edu.gh 126 TABLE 35 Atmospheric Relative Humi~ity of Cocoa Farm at Tafo, Ghana (l5-year period. 1938 - 1952). Data from urquhart (1955) }ot)nth Mean '7. R.H. Mean 1. R.H. 9.00 a.m. 3.00 p.m. January 85.9 54.1 February 83.7 51,3 March 81.6 54.7 April 80,S 57.9 May 81.7 62.3 June 84.7 67.9 July 86.0 68.9 Auguat 85.9 65.3 September 85.1 70.6 October 83,1 69.8 November B1.B 65.4 December 84.2 61.4 University of Ghana http://ugspace.ug.edu.gh 127 The effect of desiccation on the sporangia was examined by incubating the sporangia on glas8 lids of solid vetch glasses at humidities of 30 to lOOl R.H. (see ~terials and General Methods 1i (0) and (p) for varying periods. and then germinating them in distilled vater at 22°C. The sporangia were extremely sensitive to desiccation. More than 10 tests were carried out to confirm the -results obtained. The sporangia were k1lled by exposure to all the humidities tested. lO, 40, 50, 60, 70, 80, 90 and 951. R.H. (Table 1) for five minutes. No viable sporangium was a180 found after exposure for only one minute to humidities between lO and ~. R.H. HIlny of the .poran- 8ia 8urvived exposure of 957. R.B. for one minute, and they were used to determine the longevity of desiccated sporangia of the three size-classes. Tbe data in Table 36 for sporangia of the Small, Medium and Large size-classes were calculated from a total of 90, 512 and 164 sporangia, respectively. Larger sporangia apparently .uffered greater stress on exposure and lost viability more quickly than smaller sporangia. When no germination occurred after drying for five minutes the sporangia were severely indented and on re-wetting the cytoplasm did not refill the sporangium (Fig. 10). University of Ghana http://ugspace.ug.edu.gh 128 Percentage of P. palmivora of the three size-classes viable after storage at 2SoC at 95% R.H. for one minute. Sporangial size-class Percentage germination (baaed on sporangial (Percentage viability) after length) incubation in distilled water for 2 hours -------------- Small 37.7 « 24.S \.lID) Medium 30.6 (24.5 - 48.5 \.1m) Large 32.9 (> 48.5 lJD) University of Ghana http://ugspace.ug.edu.gh (.J;*~1!; ., •••: ••.:! ••, 0- :::! SOfJm FIG.10 CAMERA LUCIDA DRAWING TO SHOW SICKLE - SHAPED CYTOPLASM OF SPORANGIA OF P. PALMIVORA DESICCATED AT 95% R.H. FOR FIVE MINUTES AND THEN PLACED IN DISTILLED WATER. University of Ghana http://ugspace.ug.edu.gh 130 v. GENERAL DISC1.5SI0N Numeroua fungal para.ite. attack the cocoa tree througbout all areas where it is grown. The most important, both because of its world-wide distribution and the magnitude of the 10S8es it cauaes. i8 without doubt Awtophthora palmivora. It can infeet the roots, stems. leave. aod pod8 of cocoa. Of most concern to the cocoa industry is the direct reduction of harve8table crop that arises from pod infection, blackpod. In extensive spray trials with 28 sitea in Ghana during 1954-S6, Bowden (1961) had 35 -41 per cent black pOd in una prayed plote, about half on ripe and half on unripe pods. This would put actual 108ses cloae to 20 per cent. Efforts to produce heavier yield. to offset this 10S8 have not always been successful. Oech8li (1957) and Thorold (19S9) e'pecially have emphasized that the higher the yields, the higher the incidence of black pod. Effective control of Phytophthora pod rot has unfortuna- tely eluded all cocoa growing countries throughout the years. A very thorough knowledge of the biology of P. palmivora is necessary as a firm scientific foundation for the formulation of control measures. The object of the present study vaa chiefly to determine the lerminatlon capacity and phYSiology of different-sized -:~-=~vora, an aspect of the biology of the University of Ghana http://ugspace.ug.edu.gh 131 fungua hitherto completely ignored in the various germination studies so far made. It would be possible then to evaluate the pathogenic power of inoculum of p. palmivora consi&tina mainly of sporangia of a particular size. The sizes of aporan- gia formed on some clones and culture media were also investi- gated. Polymorphic sporangia appear to be a notable feature of many Phytophthora speCies. In P. palmivora the sporangia vary mainly in size and not shape, although some of the very large sporangia tend to assume a cylindrical instead of the typical pyriform shape (see Pig.2). In exceptional ca.es. the variation AmonS" the sporangia includes shape a8 well as size. Zentmyer and Jefferson (1974) found that the majority of sporangia of an isolate of Phytophthora citricola sawada obtained from avocado were regular in 'shape. ovoid to obpyrUorm (40 - 55 um in length) Bome slightly flattened on one side, or curved near the apex. In addition to regular sporangia the fungus charac- teristically produced a Wide variety of irregularly shaped 'porangia. Many unusual shapes were produced. These ranged from forma with two apices, shallowly or deeply bifurcate. sometimes unequally so, to more striking conspicuously large types (up to 250 \J1D in largest dimension). more irregular in .hape with tbree to fauf branches (lobes) variously dl'po.ed. ~~~nched sporangia were functional, liberating University of Ghana http://ugspace.ug.edu.gh 132 zoo'pores through their exit pores. Studies were not carried out epeclflcally to compare the physiology of the typical and the irregularly ahaped sporangia. Th~ physiology of the different morphological forllW of sporangia produced by ghytophthora capsici, Phytophthora mexicana, Phytophthora omnivora and Phytophthora phaseoll on the different host plants in the investigations of Leonian (1927) wae also never studied. The results of the present work showed that P. ealm1vora formed sporangia of different sizes on all media te8ted and on pods of four cocos elones. The lengths of sporangia from pods of Amalonado cocoa ranged from 12.5 to 60.5 ~m (see Table 2; Pig.2) of which approximately 71 per cent were medium-eized (24.5 - 48.5 ~m in length) and 9 and 20 per cent, re.pectively, were small-.tzed (less than 24.5 ~m in length) and large-sized (more than 48.5 ~m in length) (see Table 13). These rang.s of sporan- glal length were adopted In this work to represent three size- classes: Small (small-slzed), Medium (median-sized) and Large (large-sized). The widths of the sporangia were not directly related to the lengths (see Pig.l) and could not therefore be ueed for the separation of the sporangia into size-classes. There was an organised di.tributlon of the sporangial types on the sporangiophore. Larger sporangia were charaeterietleally 6porangiophore and the smaller sporangia \ University of Ghana http://ugspace.ug.edu.gh 133 appeared last (see Fig." piate 6). !he data in Table 18 showed that among SO .porangia formed first by the sporangio- phores, 82 per cent were medium-sized aDd 18 per cent 1erge- sized. None belonged to the small-size class. The proportion of the small-sized sporangia then increased as more sporangia were produced. An assessment of size of sporangia siath from the base of the sympodium showed 71.4 per cent small-sized sporangia and 28.6 per cent medium-sized. Very few species of fun8i with polymorphic spores show a distinct pattern of spore development. In £!!!osporium ~­ !!!! the opposite condition is found (Hughes 1953). Tremendous- 1y long, branched, acropetal chains composed of more or lees lemon-shaped amero8PQI:'e8 ' are formed. Towards the bale of the chains the conidia are longer, almost cylindrical. and merge gradus1ly with the thin, branched conidiophorel below as they do with the shorter conidia above. These two contrasting processes argue for different mechaniSms for spore development • .~ ~ has been shown by Wang and Bartnicki-Garcia (1973) to employ as storage polysaccharide, a-I. 3-g1ueanl similar to algal 1aminaram (Pbaeophyta and Chrysophyta) for which the designation myco1amioaran has been proposed (Wang and Bartnicki-Garcia. 1974). These highly soluble glucans were first isolated from the mycelium of Phytophthora cinnamon1 (Zevenhuizen anrl ~·tnicki-Garc1a. 1970) and later found 1n ' .. ' r~~, ~teB (Faro, 1972s,b). A detailed charac- University of Ghana http://ugspace.ug.edu.gh 134 terlzation of the molecular structure of mycolaminaram8 from p, palm1vora revealed that these were 11 polymers (lIP 29-36), Slll8 made exclusively of glucose units, with the main chain linked by 6-1, 3 bonds and one or two branches at C-6; the number of branches and the molecular size depend on the developmental stage of the fungus (wang and Bartnicki-Garcia, 1974). Whereas in the vegetative mycelium of Phyt0phthora all mycolaminaran molecules are neutral, in the sporangia and in the zoospores, most of the molecules become phosphorylated (wang sod Bartnicki- Garcia, 1973). During vegetative mycelium development, cyto- plaSmic mycolaminaran i. synthesized (33 per cent dry weight). When the fungus sporulates it mobilizes massive quantities of cytoplasm into the expanding sporangia. Very likely, the vast reserves of mycelial mycolaminaran are translocated into the sporangia where they are available to support development. They may be the main or even the sale source of glucose to manufacture the sporangial wall aad fulfil other metabolic needs, It could be envisaged that hyphae supporting the spa- rangiophores become gradually depleted of mycolaminaran and thus limiting the size of sporangia formed last on the sporan- giophore. A parallel phenomenon involving the 8torage of trEhalose has been described in other ~ungi and i8 considered to function .. ,-"'. ~.:.:~ of energy ~ndel. Vitols and parrish, 1965; University of Ghana http://ugspace.ug.edu.gh us Horlkoshi and Ikeda, 1966). The accumulation of trehalose in the vegetative mycelium of ~:~spora ~s~ Sheer and Dodge 1s 81gnificantly affected by eonid/ation. Following the initia- tion of conldiation there is 8 rapid decrease in mycelial trehalose content with a sharp increase in the concentration of the enzyme trebal~se (a,a'-glucoside l-glucohydro1ase; EC3.2.1.28) which hydrolyses trehalose. High levels of both trehalose and trehalase are present in newly formed conidia (Hanks and Sussman. 1969). The range of sporangial size found here was greater than that reported by Tarjot (1974). He noted sporangial lengths of 3S to 50 ltlD and widths from 23 to 28 um. The widths recor- ded in the present studies ranged from 15 to 30 J.l m. A tenta- tlva explanation for the narrower range obtained by Tarjot (1974) is that different strains of P. pelmivora had been used. The existence of different physiologiesl activities among strains of same species of fungus is not unknown. It has very recently been proposed by Zentmyer. Kaosiri and Idosu (1977) that four distinct groups of ~1m1~ exist based on characteristics of stalk or pedicel of the sporangia. The four groups are: I Sporangial stalk short and thick. less than 5 1I m in length (Morphological Form I). II Sporangial stalk of intermediate length and rather -z ~r~ (Morphological Porm II). University of Ghana http://ugspace.ug.edu.gh 136 III Sporangial stalk very long and moderately thick; more than 15 ~m in length and up to 250 ~m. Sporangia with these long stalks also have a high ratio of length/breadth (usualiy over 2) (Hbrphotogicai Form III). IV The sporangia are not caducous, and therefore really not typical of the species (It>rphological Form IV). It is useful to record that the isolate used in this work belongs to the MOrphological Form (MF) I (see Fig.5). Continued studies in relation to sizes of sporangia formed by P. palmivora on culture media supported the view of Tarjot (1974) that the production of sporangia on nutrient agar is highly variable. His report did not contain details of the observations made and a comparison with findings here cannot be made. Although the shape of the sporangia was generally not altered, the proportions of the amell-sized, medium-sized and large-sized sporangia varied with the culture media (substrate). Cassava-dextrose agar gave the most drama- tic effect. As much as 61.2 per cent of the sporangia were small-sized and only 1.8 per cent belonged to the large size- class (see Table 4a). On the remaining media, the percentage of the mediBm-s1ze sporangia was greater than either of the ~o sile-classes. Tbe 8ubstrate consisted both of na~ura1· 8gar media, Milt-dextrose agar Potato-dextros2 agar aDd ~am- University of Ghana http://ugspace.ug.edu.gh 137 dextrose agar and synthetic agar media containing Fructose, Galactose, Glucose and Sucrose as carbon sources and Ammonium tartrate, Asparagine, Peptone and Sodium nitrate as nitrogen source. It would naturally be impossible to find a substrate that would entirely encourage the formation of sporangia of only one size-class, The influence of any substrate would rather be recognised as supporting the production of sporangia whose lengths would provide a particular type of distribution curve. A normal distribution curve would show a distinct peak in the centre of the range of the medium size-class and two other posBible curves would be skewed either to the left (towards small size-class end of the range) or to the right (towards large size-class end of the range). Normal distribu- tion was obtained with sporangia on Potato-dutroae agar (see Table 4c) and on media containing Peptone and Sodium nitrate (Bee Tables 8 and 12). Length distribution curves skewed to the left were formed by sporangia produced on Cassava-dextrose agar, Halt-dextrose agar, yam-dextrose agar (see Tables 4a,b, and c) and on medium containing Ammonium tartrate as Nitrogen source. Galactose, Glucose and Asparagine media induced the formation of sporangia that produced length distribution curves skewed to the right (see Tables 6, 10 and 12). This last group of media had the same effect on sporangial size a8 the cocoa clon~a ~'~~do. T8S/199. 163/967 and T79/467 (8ee Tables 13- ~-~"ng to note that all the Cocoa clones University of Ghana http://ugspace.ug.edu.gh 138 favou~ed gene~a1ly the formation of larger sporangia which the present studies have shown to be more vigorous than the smaller sporangia, The auitability of cocoa pods for the production of large sporangia could be due to either the presence of some important compound a which most of the culture media lacked or a~ evolutionary adaptation of P. palmivora to its bost tiSsue. It would be useful to carry out similar investigations with other host species io any future studies. Ao important feature that should always be considered in such studies is not only the preponderance of either larger or smaller sporangia in the population but also the amount of sporangia produced. A very - large population of less vigorous sporangia might be found to be u1timBte1y more dallllSging a. disease inoculum than a very small population of very vigorous sporangia. Degree of sporulation on the various culture med1e varied extensively. Sporulation waa practically uniform on media containing the following concentrations of Glucose: 1.0, 2.0, 3.0. 4.0 and S.O g/t. The densities of suspensions prepared with stan- dard discs of sporulating cu1turea ranged from 60 x 103 to 68 x 103 sporangia per mI. suspending medium. Glucose wes by far a 8uperior medium for sporangial production to Fructose. Galactose and Sucrose which gave the respective densities of 12 x 103 and 24 x 103 sporangia per 1111 suspending medium. University of Ghana http://ugspace.ug.edu.gh 139 Among the nitrogenous ~ompounds tested. sodium nitrate was the most suitable for sporangia1 production. The standard 3 teat for sporangia1 density gave 128 x 10 sporangia per m1 3 3 3 s"..pending medium in comparison 76 x 10 , 40 x 10 and 16 x 10 aporangia per m1 suspending medium on Asparagine, Peptone and Ammonium tartrate media, respectively. Variation also occurred among the natural media, cassava- dextrose, Ml1t-dextrose. Potato-dextrose and Yam-dextrose agar where the following densities were obtained. 40 x 103• 4 x 103 • 4 x 103, and 36 x 103 sporangia per m1 8uspendiog medium, respectively. In theae cases it was not possible to attribute the differences obtained to any specific factor., since the medium contained complex natural products. There was no specific pattern of respenae in common with other fung_l species. Response was specific to each compound. The influence of other compounds that may playa role in sporulating P. palmivora in the field cannot be inferred from theae or other studies, The effect of each compound must be specifically investigated. The importance of the substrate would be further enhanced if high aporulation is accompanied by fast vegetative growth as was observed on Cassava-dextrose agar in comparison with media '~ubohydrates (see Tables 5 and 9), Among the \ University of Ghana http://ugspace.ug.edu.gh 140 nitrogenous compounds, Asparagine encouraged best the produc- tion of large-sized sporangia (see Table 12). The culture on sodium nitrate medium, however. supported such a profuse amount of sporangia (128 x lo.31ml suspending medium) that it would turn out to be a better source of inoculum than Asparagine with spo- rulating density of 76 x 103 sporangia Iml suspending medium. The results showed that different proportions of sporangia from the three size-classes were capable of germinating indirec- tly, by means of zoospores, in distilled water. Medium -sized and large-sized sporangia germinated very well at the optimum temperature (220 C) attainlng germination of 89.7 and 96.0 per cent, respectively (see Table 19). The rate of germination of the medium-sized sporangia was, however, conaiderably falter than that of the large-dzed 'porangia. Thus, after one hour the percentage germination in the former was 64.2 and in the latter 38.2 per cent. The reserve nutrients, most probably lipids and phosphoglucans, might be closely sim1lar in concen- tration in sporangia of the two size classes since final levels of germination were very close. The smaller cytoplalm of the medium-sized sporangia was apparently more quickly 'saturated' in the suspension and initiated germination processes earlier. The comparatively lower germination, 72.1 per cent, in the lmall-abed sporangia was. on the other hand, mere Ukely to be due to low level of reserve nutrients. This theory i. supported University of Ghana http://ugspace.ug.edu.gh ~l by the results of the three exp~riments which examined the number of germ tubes formed by sporangia of each size-class in water at 30oe: the maximum growth attained by germ tubes of sporangia of different size-classes supported by the endogenous substrates and studies on the ability of sporangia of different sizea to form secondary sporangia. Very few germ tubes were formed by the small-sized sporan- gia, and only 8.7 per cent had more than one germ tube. Two or more germ tubes emerged from 29.6 and 31.9 per cent of the germi- nated median-sized snd large-sized sporangia Clee Table 22). The germ tubes of sporangia of these two size-classes achieved respective mean max~ lengths of 1449.2 and 1408.0 ~m whil.t the mean maximum length of germ tubes of the small-size sporan- gia was l278.0~m (see Table 23: Fig.6). It therefore appears that in nature, the greater nutrient reserve in the larger sporangia not only leads to the mobilisation of a larger number of infective units in the host's environment. but it also accelerates their growth rate, and presumably, therefore their likelihood of infecting in a given period of time. Again, sporangia of the medium and large size-classes were able to form. on garmination. secondary sporangia in greater number. than lporangia of the small size-class. The percentages of germinated sporangia incubated at lOoe in the mediu~ and large 81ae-c18sses University of Ghana http://ugspace.ug.edu.gh 142 With secondary sporangia were very cimilar, 54.0 and 53.4 p~r cent, respectively. The value fot the small-sized sporangia was 43.3 per cent (see Table 24). Secondary sporangia formed by Phytophthora species were always smaller than the primary sporangia bearing them (Waterhouse, 1954). Germinating sporangia of P. palmivora also formed smaller secondary sporangia (sea Table 25j plate 9), and the sizes recorded in this work ranged from e mean length of 21.0 \1m for secondary sporangia of the small size-class to 32.4 \1m for those of the large size-class. Since evidence from this work has shown that very small sporangia were le8s vigorous comparatively, in- fectivity of the secondary sporangia formed by sporangia of tbe small and medium size-classes would most likely be low. It is reasonable to assume that development of the secondary sporangium was supported by endogenous substrates in the primary sporangia. There was a marked difference between the mean lengths of the secondary sporangia formed by median-sized and large-sized sporangia which had been found judged by their germinating capacities to contain almost the same amount of nutrient reserves. This indicates tbat otber factors, apart from amount of reserve nutrient, might be operating in deter- mining the sue of the secondary sporangium. The number of zoospores formed in a sporangium was ~o the size of the sporangium. The large- University of Ghana http://ugspace.ug.edu.gh 143 sized sporangia formed on the average 19 zoospores per sporangl~ and the range was 17 to 25 zoospores. The number was far greater than the mean of 9 zoospores in a sporangium of small size-class. In this group. the highest number found in a sporangium was 12. Mediulll-.tzed -sporangia produced on the average 15 zoospores. The zoospores of the large-sized sporangia were, further more lignificantly larger (10.3 ~m diameter when encysted) than zoo- spores of the other sporangia (8.9 - 9.0 jJ m diameter when encysted) $ee Table 20). The conditions under which the zoospores were formed did not allow further observations on the habits of the zoospores of each type of sporangium. It was possible that varia- tion in behaviour might exist. Dukes and Apple (1962) demonstra- ted dissimilarities in the habits of zoospores of virulent and weakly virulent isolates of ~~thora parasitica var. nicotia- ~ Tucker. Zoospores of virulent isolates had a considerably longer motility period than those produced by weakly virulent or intermediate types. The average motility period for the virulent isolates was 12 -15 hours whilst weakly virulent types had an average motility period of only 5 - 8 hours. Clearly, zoospore formation in f. palmivora did not need any chemical stimulation and near 100 per cent indirect germina- tion occurred in distilled water at the optimum temperature. Sporangia germinating at 30°C in distilled water by means of University of Ghana http://ugspace.ug.edu.gh 144 germ tubes were not that successful and only 7.0 -1.6 per cent of the sporangia of all size-classes could produce germ tubes (see Table 21). Ultrastructural studies of the directly germi- nating sporangia of P. palmivora by Clerk (1974) and Hemmes and Hohl (1973) showed that the sporangia always contained flagella at maturity. One of the first steps in direct germination was the reversal of elements needed for zoosporogenesis accompanied by elimination of the unwanted flagella. '!'he breakdown of the flagella involved two main stages: first, there was cleavage of the ensheathing vacuole of the flagella into small vesicles leaving the axoneme unenclosed in the cytoplasm. Later these vesicles dispersed in the cytoplasm. In the second stage the axonemes gradually disintegrated involving gradual loosening of the parallel alignment of the microtubules and then a depolyme- rization of the microtubules. At approximately the same time, a germination wall is laid down in apposition to the sporangial wall. The germ tube emerged by penetrating the sporangial wall and was surrounded by the extensible newly formed germination wall. Direct germination in distilled water was poor probably because many of the sporangia did not have adequate energy source for the series of complex events that lead to germ tube formation and needed the support of extraneous nutrients or energy source. Such support was given by many different compounds which ~-'Js_,. -~;ne lOW I.i'rect germilUltion (7.0 - 7.6 per cent) to University of Ghana http://ugspace.ug.edu.gh 145 even 100 per cent in certain instonce3. An abstract of the relevant data showing the highest direct germination acbieved in each test is provided in Table 37. ~e unspecific stimula- tion is not easily understood, for it implies that the metabolic processes of the sporangium can be affected at widely separated points. Por example,vit¥lm!n free casein hydrolysate presumably acts through N-metabol1sm, Yeast-extt"act either acts like casein hydrolysate or as a source of vitamins, perhaps involved in enzyme synthesis or activity and (NH4)P04 presumably as co- factors in some enzyme systems. The carbohydrates in the cocoa pod-extract and root exudate would very likely act through respiratory pathways. It would be observed that the large-sized sporangia always be~fitted best by the addition of nutrients, pointing to the existence of an already greater amount of internal reserves. When the sporangia were stained for lipide and proteins, it was found that the densities of each substance in sporangia of different sizes were the same. Since a larger sporangium provides a bigger storage space,a greater amount of nutrient reserves would be deposited there, The concentration of the_carbohydrate reserves, phosphoglucans, was not determined and it is not possible to assess their contribution to the diffe- renees in response to externally supplied nutrients. Staining for phosphoglucans was not carried out because the large amount of the glucans in the sporangial wall (25 per cent of cell wall) fhe Btain. University of Ghana http://ugspace.ug.edu.gh 146 Abstract of o.ta on SdJDulat.ed Direct Genl1DBtion (Tab1e& 26, 27. 30. 31, 32 and 34) in sporangia of P. palmivora at 30°C. (Direct Germination in Distilled Water per cent) Sporangia 1 size-class Best Stimulation of (based on Stimulated Gerudna tion by sporangia 1 % Germination length) 100 Cocoa pod extract (I.Og/2OmI H2O) 21.7 Cocoa root exudate (Undiluted) . Small 56.8 Casein hydrolysate (1:01. : w/v) « 24.5 lJm) 49.2 peptone (1.<14 : w/v) 79.2 Yeast-extract (1.0'!. : w/v) 34.0 (NHt.)2HP04 (2.0 mM) ---- lOa Cocoa pod extract (0.5 - 1.Og/20m1 H2O) U.8 Cocoa root exudate (1:1 dilution) Mediwn 55.1 Casein hydrolysate (1.07. : w/v) (24.5 - 48.5 \.1m) 54.0 Peptone (1.0'!. : w/v) 85.9 Yeast-extract (1.07. : w/v) 34.8 CN!\)2HP04 (2.0 mM) 100 Cocoa pod extract (0.5 - 1.0g/2Om1 ~O) 34.0 Cocoa root exudate (Undiluted) Large 77 .9 Casein hydrolysate (1.0'1. : w/v) (> 48.5 lJm) 55.0 Peptone (1.0% : w/v) 89.2 Yeast-extract (1~0% : w/v) "".0 (NH4 )2HP04 (2.0 mM) University of Ghana http://ugspace.ug.edu.gh 147 It i. considered that information available from the.e atudies lhould be extended in future investigations before an attempt il made to explain the response to stimulatory substan- ce. on the basis of nature of the functioning or internal phy- siological mechanisms of sporangia of different sizes. At least. it hal been .hown here that mitochondria (.ee flate 13) and RNl concentrations are uniform in all the 8porangia. The larger .poraogia germinating directly in nutrient .olutioll8 were liable to produce numeroUS germ tube.. "l'be amall-sized sporangia in solutions of 1.0 per cent calein hydro- lysate formed more germ tubes. It was po •• ible that the concen- tration was optimal for theae sporangia but not for the others (lee Fig. 9). 'J.'he exudates of roots of many plaQU have been analyeed by many worker.. nae .pactra of compounds. elpec1ally carbohydrates and amino acids. reported for the different plante vary conside- rably; but comparisons between plants lII8y be misleading because of the different conditions under which the experiments were conducted by different investigators. Even the exudation patterns for a siogle plant .pedes such as wheat (Triticum ~ Lam.) as found by four different workers varied conside- rably. Katznelson. Rouatt and Payne (19SS) identified only five amino acids in the exudate. Riviere (1960). Tesar and Kutacek Sncura and H$cura (1962) identified many more University of Ghana http://ugspace.ug.edu.gh 148 amino acids, 11. 13 and 15 respectively. Cocoa root exudate (see Tables 28 and 29; Fig.7) is omang the most complex report- ed for flowering plants so far. Whereas it would be possible to reproduce the extent of stimulation provided by the cocoa pod extract in laboratory experiments in wounds created in the pods in the field through rodent and insect attack. the influence of root exudate on spore germinstion is critically dependent on other factors lQ· the soil environment, and the degree of stimulation described in this thesis are valid only for the conditions under which tbey were established. The most important factors of the soil that may modify the response of the sporangia to the exudate are soli pH, inorganic ions and general soil fungistasis. CaCl2 lowered indirect germination and totally inhibited direct germination, and (~)2HP04 reduced the level of indirect germination (see Tables 33 and 34). Although zoospore formation in the small-sized sporangia was more severely affected than~tbe larger sporangia, it is unlikely that tbe latter would be pro- tected from fungicides currently in use, since the recommended concentrations are definitely lethal to all the sporangia. This habit would enable the larger sporangia to withstand other less potent substances such as antibiotic substances and inhibitory compounds such 8S orsenic acids in root exudates. Some fungi are extremely sensitive to desiccation. Viabili- . \of powdery mildews and sporangia of Oomycetes University of Ghana http://ugspace.ug.edu.gh 149 is best preserved in wet conditions as in the case of conidia of EryBiphe gaminis de Candolle Co"!: Merat (!'~tzger, 1942), Erysiphe ~ de candolle ex Saint -Amana (Yarwood, Sidky, Cohem and Santilli, 1954), Phyllactinia ~le3 (pers.) Karst. (Ankara, 1968)and ~tophthora infestans (Warren and Colhoun, 1975) P. palmivora belongs to this category. Humidity was found to be a factor powerfully influencing survival. Sporangia at 95% R.H. lost viability in 5 minutes. Quick death at 90'l. R.H. and below was accompanied by other events. The thin walls of the sporangia collapsed as water was lost in air. These structural changes that occurred in the shrunken protoplast were irreversible. Shrunken sporangia floated in water, pushed back the infolded sporangial wall but the protoplast remained sickle-shaped (see Fig.lO). Vast quantities of ~~~ are likely to die immediately on production since the atmospheric humidity of the cocoa farm, even under the canopy, never reaches 907. R.H. (see Table 35). It is, however. possible that transpiring cocoa pods would keep the atmosphere around the sporangia saturated with water vapour. Greater proportion of the sporangia might therefore remain viable than would have been expected. In the light of the present results it is difficult to imagine that sporangia can be transported for considerable distances and remain viable if they have been exposed to drying during transportation. University of Ghana http://ugspace.ug.edu.gh 150 In all the germin3tion e:.perimentD. either during indirect or direct germination. no germin~tion C7er ocr.urred within five minutes. The ability of the small-sized sporangia to survive better, at least in one minute, than the larger sporangia i8, therefore. not a valuable characteristi~. For, they all would be dead long before germination processes could be initiated. An important aspect of the biology of ~~~. a serious pathogen that continues relentlessly to take heavy toll on a vital economic crop. cocoa, has been studied. The way in which the proportion of sporangia of different sizes in the spore population may be altered by media and cocoa clones has been identified and the germination capacity of sporangia of different sizes bas been defined. A very important finding here is that larger sporangia are more vigorous and are more re8istanl to inhibitory substances than smaller sporangia. In connection with the spread of black pod, the importance of cocoa clones and other plant host species will among other things depend on type and amount of sporangia, the major infection units, they would produce. It is suggested that the type of spo~angia supported should, in future. be included among the criteria used for the assessment of the suitability of sny newly bred cocoa clone. University of Ghana http://ugspace.ug.edu.gh 151 SUMMARY 1. The sporsngitl ..•o f P. palmivo~3 were of vc:::ying sizes. with lengths ranging from 12.5 - 1.5.5 1.I m to 57.5 - 60.5 1.I m. 2. Variation in sporangial widths t-7as not very great. 1.5 - 30um. and the width was not closely related to length of the sporan- gium. 3. For experimental purposes. the sporangia were categorised into thre~ size·classes based on sporangial length. (8) Small size-class less than 24.5 l.Im (b) Medium size-class from 24.5 to 48.5 ~m (c) Large size-class more than 48.5 l.Im. 4. On Amelonado cocoa, the myceUum formed 8.7. 87.1 and 4.2 per cent small-sized, median-sized and large-sized sporangia, res- pectively. 5. There was a recognisable pattern of formation of the different- sized sporangia; larger sporangia were generally formed first by the sporangiophore and the smallest sporangia formed last. 6. The proportion of sporangia in the three size·classes was different for the different substrates. The respective percen- tages of sporangia in the Small, Mediu. and Large-size-classes on the different nutrient media were: University of Ghana http://ugspace.ug.edu.gh 152 37.0 and 1.8 jtSIS UNIVERSITY of coIlANA. PI1~IIQcti"ia cor!!lea (~rs.) Kars, . c.' 1 ARAGAKI, M., H:>BLEY, R.D. and HINE, R.B. (1967). Sporangia germination of Phytophthora from Papaya. M"«:OLOGIA, ll: 93 ~ 102. ASOHlNING, E.J.A. (1964). Black pod disease. Boot infection of cocoa by ~~ Butl. Studies on varietal resistance. Rep. Cocoa Res, Inst. Ghana. Acad. SCi •• 1962 - 63. pp. 23 - 25. BARtON, R. (1957). Germination of oospores of PYthium m81111liUetum in response to exudate from living seedlings, MTURE (London) 180. 613. BIHPONG, Christina E. (1969). Studies on factors influencing zoospore motility and chemotaxi8 in Phytophthora palmivora (Butl.) Butl. H.Sc. thesiS. University of IR ISS. DRECHSLER. C. (1947). A Basidiobolus producing elongated secondary conidia with adhesive beak• • BULL. TORREY BOT. CWB 1i. 403 - 413. "'\ Il.W species of Conicliobolus otcur,in,9 In leaf mo Id . -~. 4 0 , 104 - 115. University of Ghana http://ugspace.ug.edu.gh 162 DRECHSLER. C. (1954). Two species of Con1diobolus with minutely ridged zygosporcs. AMER. J. BOT. 41. 567 - 575. DRECHSLER. C. (19558), Three new species of conidiobolus isolate from decaying plant detritus. AMER. J. BOT. 42, 437 - 443. DRECHSLER, c, (l955b). Two new species of Conidiobolus that produce microconidia. AMER. J. BOT. 42, 193 - 802. DRECHSLER, c. (1960). Two new species of Conidiobolus found in plant detritus. AMER. J. BOT. 47. 368 - 377. DRING. V.J. (1958). Conidiobo1us rhysosporna Drechsler in Great Britain. TRANS. BRIT. MYCOL. SOC. 41. 52 - 54. DUKES, P.D. and APPLE. J.L. (1962). Relationship of zoospore production potential and zoospore motility with virulence in Phytophthora parasitica var. nicotianae PHYTOPATHOLOGY 62, 191 - 193. EBBEN, N.H. and WILLIAMS. P.R. (1956). Brown root rot of tomatoes. I. The Associated fungal flora. ANN. APPL. BIOL. 44, 425 - 436. ELSNER. P.R., HORTO~J J.C. and BOWEN, C.C. (1970). Fine structure of sporangia of Phytopbthora infestans during sporangial differe~tiation and germination. - - .\ BOT. ~ 1765 - 1772. University of Ghana http://ugspace.ug.edu.gh 163 PARO, S. (1972a). A soluble beta-l,3-g1ucan found in selected genera of oomycetes. J. mm. MICROBioL. li, 393 - 394. PARO. s. (1972b). ~e role of a cytopiasmic glucan during morphogenesis of sex orgQn~ ~n A~hly3. AMER. J. BOT. ~. 919 - 923. HALM, M. (1970). Studies and some aspect3 of germination of sporangia of Phytophthora pa1mivora (But1.) Butl. B.~c. HO~~. D!SSERtA~ION. University of Ghana, Legon, p.72. 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COCOA pp.230 Longmans, Green London. VRANY, J •• VANCURA, V. and ~C~> J. (1962). the effects of foliar application of some readily metabolized substances, growth regulators snd antibiotics on rhizosphere microf10ra. fROBIOL. 2, 61 - 70. University of Ghana http://ugspace.ug.edu.gh 169 ~NG, M.C. and BARTNICKI-GARCIA, S. (1973). Novel phosphog1ucans from the cytoplasm of Phvtophthora p~lmivora and their selective occurrence in cert~in life cycle stage• • J. BIOL. CHEM. 248, 4112 - 4118. ~NG, ~C. and BARTNICKI-GARCIA~ s. (1974). Mycolaminarans: .torage B-1,3-D-g1ucans from the cytoplaem of the fungus Phytophthora palmivor~. CARBO~lI. RES. ll, 331 - 338. WARREN, R.C. and COLHOUN, J. (1975). Viability of sl'Orangia of Phytophthora infestans in relation to drying. tRANS. BRIT. M'LCOL. SCC. §i, 73-78. WATERHOUSE, G.M. (1956). The genus Phytophthora.Diagnoses (or Descriptions) and Figures from the original Papers. THE COMMJNWEALnl MYCOLOGICAL INSTITUTE, KEW, SURREY, 120 PP. WATERHOUSE, G.M. (1974). EhY.to~~thora palmivora and some related species. IN PR'll'OPHnlORA DISEASES OF CCCaA., ED. P.R. GREGORY, LONDON. REP. W. AFR. CCCOl\ RES. INST., 1958 -59, pp.26 -27. WHARTON, A.L. (1961). Blnck pod disease; Resistance and tolerance. REP. W. AFR. COCOA Rr.:~. nlST., 1959 - 60, p.2S. University of Ghana http://ugspace.ug.edu.gh 170 WHARTON, A.L. (1962). Black pod disease. Spraying trials against Phytophthora palmivora Butl. Resistance and tolerance. REP. W. AFR. COCM RES. INST •• 1960 - 61. pp.26 - 29. WHITE, N.H. (1954). The use of decay crops in the eradication of certain soil borne plant diseases. AUSTRALIAN J. SCI. 17_, 18 -19. WILLlA~, W.T. and WEBSTER, R.K. (1970). Electron microscopy of the sporangium of Phytophthora capsici. CAN. J. BOT. 48. 221 - 227. WOOD. r.A •• SINGH. R.I'. and HODGSON, W.A. (1971). Characterization of A virus-inhibiting polySAccharide from Phyt0pbthora infestans. PHYTOPATHOLOGY g, 1006 - 1009, YARWOOD, C.E •• SIDleY, S •• COHEM, M. and SAm'ILLI. V. (1954). Temperature relations of powdery mildews. HILGARDIA ;'E, 603 - 622. ZENTHYER, G.A. and THORN, W.A. (1967). Hosts of Phytophthora cinnamoni. CALlP'. AVOCADO SOC. YB.g. 177 -186. ZEN'l'MYER, G.A. and JEFFERSON, L. (1974). Studies of A!ytophthora citricola isolated from ~ americana. MYCOLOGIA~, alo - a4S. University of Ghana http://ugspace.ug.edu.gh 171 ZENrM!ER, G.A., ~OSIRI, T. and IDOSU, G. (1977). Morphological forms of Phytophthors pa1mivora, and the utility of sporangium stalk length as a Taxonomic character. PHYTOPH'lliORA NEWSLETl'ER No.5, So - 51. ZEVENHUIZEN, L.P.T.K. and BARTNICKI-GARCIA, S. (1970). Structure and role of a soluble cytoplasmic glucan from Phytophthora cinnamomi. J. GEN. KICROBIOL. ll. 183 -188.