University of Ghana http://ugspace.ug.edu.gh STUDIES eN THE RELEhSE. SURVIVAL AND GmMINATION OF CONIDIA OF ~~ ~ (PERS.) KARST. A thesis presented by J OSEPH K\V~ ANKORA, B. Sc. (LRGON) in part fulfilment of the requirements ror the M.Sc. DEGREE . r· of the University of Ghana JUNE, 1968. From: The Department of Bot8l'\Y, University of Ghana, L E c;. 0 N. University of Ghana http://ugspace.ug.edu.gh ABSTRACT Conidia of ~orylea ogerminated best at 25 - 2S C. and germinated nd at the same rate at any humidity from zero to 10~ well uni fo r ml Y a f nid· erm°na.ting R.H. : but poorly in liquid water. Germ tJ.bes 0 • co l.~ ~ . l. at lower humidities were short whilst those at high ~midl.tl.es were fairly long. ApproxiJ[lB.tely 30 per cent of both germJ.n&ted. a~ ungerminated conidia shrivelled when incubated. at 0% R.H. Thl.S value 'decreased with rise in humidity to show shrinkage in only ene per cent of conidia held at 10Q% R.H. The conidia had a brief latent period of germination of 2 - 3 hours. The germinating conidia usually produced a single germ tJ.be am occasionally two. Branching of germ tubes was ra::e on glass surface but appressoria were freely formed. On the host (Carl.oa~) leaf surface the germ 1llbes grew randomly over the epidermis am equally freely formed a~pressoria. The appressoria were either terminal, intercalary or lateral. Germ tubes produced at lower humidities collapsed and shrivelled within a few hours after emergence. All germ tubes produced and held at ~ R.H. shrivelled in 11 hours and those at 80.3% R.H. in 30 hours. Those at 92, 96.9 am 100;0 R.H. showed 94.5, 25.0 am 20.2% of shrivelled germ tubes respectively in 48 hours. The Conidia germinated better in light and. produced longer germ tubes but formed fewer appressoria than those incubated in darkness. Conidia stored at 5eC. and at various relative humidities were preserved longest at the higher humidities and died quickest at the lower humidities. Longevity was however brief and did not exceed 20 days at any humidity. lulticellular, non-branching conidiophores, commonly 300 u long, arose perpendicularly from the superficial mycelium. 'Bach \lias termi- nated by a generative cell whioh formed the conidia. Usually only one mature conidium was distinguishable at the tip of the conidiophore. Immediately after maturation of the conidium the generative cell bent approximately at the middle while the two arms of the cell stood at right angles. This movement loosened the attachment of the conidium to the conidiophore and the conidium fell at the slightest distJ.rbance. The bent generative cell then abstricted the next oonidium. In still a~ oonidial chains were readily formed. A few oonidia germinated whilst ~till attached to the conidiophore. The oonidia matured predominantly l.n the dark and heaviest crop of spores were obtained in the morning. The rate of bending of the generative cell ;.'8.S the sa.me at 20 and. 0 28 C., in light and in dark and at 76 and 10Cl}o R.H. The ourvature in the generat~ve cell remained unaltered in sucrose solutions and in water. The generatl.Ve celJ,.~not phctotropir Whilst the entire conidiophore University of Ghana http://ugspace.ug.edu.gh 2 CONTENTS Pa.ge 1. INTRODUCTION. 12 II. LITERATURE REVIER III. MATERIALS AND GENERAL METHODS. 14 i. MATERIAl.s : Source and maintenance of s took culture •• 14 ii. GEtlERAL ~iETHODS: a. Humidity chambers 15 b. Maintenance of Constant Humidity 17 c. Methods of Sterilization 17 d. Preparation of conidia for germination testa 20 e. Spore germination tests 20 f. Incubation ••• 20 g. Assessment of conidium germination and germ tubG length 21 h. Experimental Precautions 22 IV. RESULTS : A. Effect of Temperature on Germination of conidia of P. coryiea 24 B. The Germinaticn of Conidia of ~orylea at various Relative Humidities ••• 33 C. Further Experiments on the Germination of conidia of p. corylaa at various Relative Humidities •• 52 a. Germination of the conidia at various relative humidities maintained with standard aqueous sal t solu tions " . 52 b. Germination of the conidia under current of air with adjusted Relative Humidities University of Ghana http://ugspace.ug.edu.gh 3 - CONTENTS (continued) ,. P8.ge D. Effect of Fluctuating Atmospheric Humidities on the germination of coniciia of F_.~ corylea at ~.trr,ospheric temperature 69 E. ]~f:C-"ct of various Relative in.:.midities on the Rate or germination of conidia of p. corylea F. Further experiments on the effect of various humi- dities on the Rate of germination of conidia nf P. corylea 82 G. Ef feet of Humidity on th·; Rate of germ tube grC'wth 88 H. Germination of conidia of .P_ __ £2!'J.l.el!: still attached to conidiophore 95 1. Survival of the germinat ,d conicciQ of p. corylea 100 J. Pattern of germination of conitlia of p. corvlea 116 K. Effect of Light on conidial germin:.. tion 131 L. Longevity of conidia of E!_..9..9.E2~ stored at different Relative Humidities 135 M. Development and Releas e of conidia of P. corylea 144 a. Development and Morphology of the C~nidiophore 147 b. Formation and Development of the Conidium 153 c. Release of ~ature Conidium 155 d. For~ation of subsequdnt conidia 165 e. Formation of conidial chains 172 f. Periodicity ~f conidium formation 179 University of Ghana http://ugspace.ug.edu.gh 4- - CONTEN'IS (con timed) P~ge N. Further observations on oonidi')phf'lre behaviour 182 a. Effect of Temperature on Bending flf the Generative Cell 183 b. Effect of Moisture on Bending of the Generativo Cell 186 c. Effect of Light on the banding of the gen"rative cell. 189 i. Ef'fect of light and dark on the berding of the genera tiv e cell 190 it. Effect of light on direction of baming of the generative call 191 V. GENERAL DISCUSSION 200 VI. SUMMARY 222 VII. ACKNOWLEDGEMBNT 227 VIII. LITmlA'lURE CITlID 228 University of Ghana http://ugspace.ug.edu.gh - .5 I. INTRODUCTION The powdery mild8ws (Brysiphaceae) are oblieate parasites which generally occur as ectoparasites and grow exclusively on the surface of the host epidermis. The species, Leveillula ~ and Phyllactinia corll~ are the only exceptions. Leveillula ~ is of particular interest because its ~celium is wholly endophytic and in like manner a portion .f the mycelium of ?hyllactinia corylea occurs bet"een the mesophyll of its rumerous hosts. Both L. taurica and P. corylea are very familiar and occur c,xtensively in he-pical and sub-tropical regions. In Ghana, and in ':{ est Africa in general, L. taurica occurs with amazing frequency on leaves of pepper (Capsicum ~ L.) and no pepper plant ever seems to escape thr, disease. p. corylea, on the other hand, has just been recently found for the first time in Gh3.M by C18rk (1966) on pawpaw (~ ~ L.) leaves. The disease ailPears as discrete patches of greyish-white, cobwebby growth on the abaxial surface of the leaf (Plates 1 and 2). Under very favourable conditions, the number of disease lesions is so great that the diseased S}ots tend to cover the entire leaf surface. Von Tubeuf (1897) and Yerwood (19.56) found that there is occasional retention of chlorophyll at mildewed spots when the entire leaf became chlorotic. In the late stages of the disease the pawpaw leav(,s Similarly be ame yellow except the sites supporting P. corylea which retained their chlor~phyll and appeared as distinct University of Ghana http://ugspace.ug.edu.gh PLATE 1: Photograph 0'( abaxial sur'(ace o'f leaves of pawpaw (C. papaya) showing white patches of mycelium 0'( p, corvlea. A. Healthy lea'(, B. Mildewed le&1'. Note the presence ot chlorophyll at early stages ot disease. University of Ghana http://ugspace.ug.edu.gh - 7 - PLATE 2: Enlarged photographs of apioes of healthy and mildewed pawpaw (e. papaya) leaves. A.. Heal thy leaf. B. Mildewed leaf. University of Ghana http://ugspace.ug.edu.gh - 8 - "green islands" on a yellow background (Plates 3 and 4). The host range o~ P. cerylea is very ,ride and the ~ungus is an almost exclusive parasite of dicotyledonous plants. For example, Cardoso (1940), Hecter (1927), Ramarishnan and Sundaran (1954), Rayss (1953), Ressencourt (1927), Rhind (1924), Wallace (1930) and Zaprometo~~ and i:ikhailof~ (1937) ~ound P. corylea on IIlllberry. The fungus occurred on~ australis (Fo~x, 1926), jasmine (Sydow and Mitter, 1933), medlar (Voglino, 1932), Lagerstroemia (West, 1933), Corylus rostrata (Miller, 1934), pear (Blumer, 1936), lilac (Viennot-Bourgin, 1944) and on Dalbergia ~~ and teak (Bagchee, 1952). Although P. corylea has, at present, been ~ound in Ghana on pawpaw alone, it is not unlikely that, ~n vie,; o~ its reported nide host range, this fungus may be parasitic on more host species in this country than hitherto rel'orted. .E:..-.corylea is certainly potentially o~ economio importance. Its significance and importance in Ghana becomes mere pro~ound with the current introduct- inn of mulberry plants into this country in connection with the silk industry. In the tropics and sub-tropics, the pov~ery mildews rarely prnduce cleistothecia and many species perpetuate themselves solely by means of co~idia (alexopoulos, 1952) and th~ conidia there~ore remain the main agents o~ infection. Conditions which therefore University of Ghana http://ugspace.ug.edu.gh 9 - PLATE 3: Photograph o~ abaxial surf'ace o~ chlorotic, mildewed leat" of pawpaw (C, papaya) at very advamed stage o~ disease. Note green "islands" o~ chlorop~ll at sites supporting P. corylea on a background o~ yellow leaf, University of Ghana http://ugspace.ug.edu.gh 10 - PLATE 4.: Photograph of adaxial lIurface of chlorotic, mildewed leaf of pawpaw (e. papaya) at very adftnced stage of disease. Note green "islands- of chlorophyll at sites supporting P. cor;ylea on a background of yellow leaf. University of Ghana http://ugspace.ug.edu.gh 11 affeot germination of the conidia of P. corylea must be thoroughly studied and well defined in view of the epidemiological implications. It is surprising that a search through the literature revealed n~ pertinent information whatsoever. This work :vas also carried ou t to provide information on another important aSPdct of the biology of P. corylea. Ressencourt (1927) and Viennct-Bourgin (1944) reported that th~ conidiophnres of p. corylea were few in number, elongated, rigid and ,lith only a single distinguishable conidium at the apex of each. Although ether workers have made similar observations and had indicated that the mature conidium fell off before the succeeding one appeared, the reason why only a single conidium occurs on a conidiophore at any time has never been investigated. The present studies were uzrlertaken to provide some information on the development, release and germination of the conidia of P. corylea;_ University of Ghana http://ugspace.ug.edu.gh 12 - II. LlTERl .. TURE REVIE.W Very surprisingly there are very few references to the biology of P. corylea in the literature. Most of the information available is in relation to host range and geographical distribution. There is no information on the effect of different factors on the germination of the conidia of this fUngus or on the pattern of conidial germination. The only information to be found in connection with tempera-rure and the fUngus is provided by Ressencourt (1927) who was infact describing a species of Phyllactinia which he thought to be at l(;3.st clesely related 1:0 P. corylea. He reported that the active d~velopment of this speci~ s was favoured by tempera-rures of 20 to 250 C. and retarded at 30oC. or over, as well as by intense damp or h8a vy rain. Foex (1926) gave a very (;)xtensive account of the development of the conidiophore and fcrmation of thG conidia of P. corylea. He wrote, "The first indication of the conidiophore was tile forma- tion of a slender cylindrical cell which rapidly elongated perpendi- cularly to the hyphae on Vihich it arose. Folloning nuclear division this cell separated into two, of which the basal cell was much longer and thinner than the apical; the protoplasm in the latter was much IllOre denser than in the former. New elements were formed by continued University of Ghana http://ugspace.ug.edu.gh 13 - division of the apical cell. Finally th~ conidiophore uas composed of a thread-like basal cell, in which the protoplasm ,7as concentrated at the top. The terminal. c ell which was the only true conidium, vias of a characteristic ovoid-conical shape." Hammarlu~ (1925), Viennot-Bourgin (1944) and Roger (1953) reported that the conidio- phore of P. coryll;a bears only a single conidium at a time. Vien=,~+'­ Bourgin, however, found that o~casicnally some cOl'Liiophorcs bore tuo conidia, whilst Hammarlund (1925) observed that E._ _c .oJ.'ylea which frequently only bear a single conidium at the ti:l) or the conidiophore, developed numerous chains in damp, still air. There is however nc information on the mechanism of release of the J;Ub.l';'; conidium. University of Ghana http://ugspace.ug.edu.gh - 14 - III. MATERIALS AND GENERAL METHOOO i. MATERIALS: The culture of P. corylea used was a local isolate obtained from a naturally infected pawpaw (Carica ~ L.) plant. The conidia of P. corylea used in these investigations were obtained from stock culture l18.intained on glass house-potted pawpaw plants. Fresh seedlings were raised every DOnth and were inoculated at the 6-leaf stage. The healthy seedlings were inoculated by gently adpressing diseased lesions of infected leaves unto the abaxial surface of the leaves. ~he temperature of the glas shouse romainod oonstant at 27 .± 1 °C. It ";ras nE:cessary to ob'\ain oonidia of uniform age throughout the work and the following procedure was adoptdd during these investigations. The leaves of pawpaw plants showing diseased lesions, which normally appear as white cobwebby patches on the abaxial surface of the leaves, were carefully tapped as a rule at 7.00 a.m. with a ruler, and a strong current of air from a bellow was blovm over th.a surfaces of the leaves to remove any !l)9.ture conidia presant. The plants were then left to allow fresh crop of conidia to develop. Conidia wer" al',mys, except where otherwise stated, removed from the leaves 48 hours after treatment, that is at 7.00 a.m. on the day of experimentation. University of Ghana http://ugspace.ug.edu.gh 15 - ii. GENERAL METHODS: (a) HUMIDITY CfLJI:BERS i) ~~ Two types of plastic boxes were u s8d as humidity chambers. They wer", either r"ctan€).llar plastic boxes (21.5 cm long, 10.5 em wide and 7.5 cm deep) (Fig. 1) or square plastic boxes (10 cm x 10 om and 7 cm deep) with tightly fitting lids soaled airtight with cellotap~. These were mostly used in germination tests requiring rddive humidities of' zero to 100j.,. The plastic boxes were found very convenient as very little condensation occurred when the atmospheric humidi W was maintained at 100% R.H. Glass slides holding conidia for germination and survival tests were sup;-;orted at the; bottom of the humidity chambers on V-piece glass rods. 1:·tatGr and solutions used for controlling the relative humidity of the atmosphere within th_ chambers were enough to cover the bottom of the boxes and rose just to midway • of the thickness of the V-piece glass rod (Fib.1). ii) Van 'rieghem Cells Solid 7/atch glasses (3.7 x 3.7 x 1.6 cms) V1iih a well 3 cms. in diameter and 1.0 cm deep werd used to serve as Van Tieghem Cells (Fig. 2) for some of' the conidium germination experiments. They were also used as rumidi ty ch<:mbers. The well of ~ach watch glass held 2 ml. of the appropriate solution University of Ghana http://ugspace.ug.edu.gh - 16 - r---·----------------------------~~LID r--------------------------------~ I ..... GLASS SLIDE /~ BEARING CONIDIA ~t"=;~~~~~-~J~§§~D]~E:::;!~~~~~OS~J_--SOLUTION TO MAINTAIN CONS- TANT RELATIVE HUMIDITY. FIG. I. RECTANGULAR PLASTIC BOX -HUMIDITY CHAMBER EMPLOYED FOR GERMINATION OF CONIDIA OF P. CORYLEA. CONIDIA =:;\=.=~::;=';:=::;:;c~;:::s . COVER GLASS . [ ~I f SOLUTION TO MAINTAIN - - CONSTANT RELATIVE HUMIDITY. '-----------------~ F IG.2. SOLID WATCH GLASS USED TO SERVE AS VAN TIEGHEM C~LL- HUMIDITY CHAMBER EMPLOYED FOR GERMINATION OF CONIDIA OF P. CORYL EA. University of Ghana http://ugspace.ug.edu.gh - 17 - to give the desired relative humidity. The top edge of the watch glass ',las lu t",d Hi th vaseline in order to form an air-tight seal ':Ihen thE: lid is placed on it. Conidia of P. coryl"a were dusted onto th~ ccntr8 of the glass lid, within a circumscribed area of 1.0 cm in diameter. The glass lid was then gently placed, with the conidium b~aring face downwards, onto tho '.latch glass. (Fig. 2). (b) MAIN'lf.N1J'ICE OF CONSTl.NT HUMIDITY Various r,lative humidities 'v7ere provid""l and maintained by eithar saturatod aqueous salt solutions, Hhich wore prepared according to the data provided by Hexler end Hasegawa (1954) (See Tabl8 1), Dr sulphuric acid solutions ~ased on th0 data of Solomon (1952) (S0e 'iable 2). Throughout the invl-stigations, norminal (f:, R.H. vias maintained with anhydrous calcium chloride and water provided 100% R.H. (c) ],E"ll!ODS OF STERILIZJ...TION Glass slid.:;s, glass covers, pippctes, flasks were all soaked in potassium dichromate for 48 hours, "lashed. ,lith detergents, rinsed under running tap and further thoroughly rinsed with distilled water. The slides and glass covers ',:ere. storJd in 9a;.; ethyl alcohol and flame-st"rilized just b"fore use. McCartn(;y tubeS which war., used in some germination tests were sterilised tog:;;tht.r ',i ith the germination liquid medium by auto- claving for 20 minutes at 15 pounds per square inch steam pressure. University of Ghana http://ugspace.ug.edu.gh - 18 - Table 1 Relative jiumidity (Percentage) provid~d by 3,:l.tur~"tE:;d Aqueous Sal t Solutions. (uata of Wexler , A. ani S. Hasegawa, 1954) Tempe- LiCI :r:4 )21-raturtl °e. H 0 NaCI SO KN03 ISS 04 in 2 1-._ . __- +-_-+_,;;.-._;-!_ --iI __ ·l~_+.--4--- o 14.7 60.6 ! 74.9! 83.7 I 97.6 99.1 5 14.0 59.<:: ~ 75.1 j 82.6 I 96.6 98.4 10 13.3 57.8 75.2 81.7 95.5 97.9 15 12.8 33.9 56.3 75.3 81.1 94.4 97.5 20 12.4 33.6 54.9 75.5 80.6 93.2 97.2 25 12.0 33.2 I 53.4 75.8 80.3 92.0 96.9 30 11.8 32.8 52.0 75.6 80.0 90.7 96.6 35 11.7 32.5 50.6 75.5 ; 79.8 89.3 96.4 University of Ghana http://ugspace.ug.edu.gh - 19 - H2S04 Solutions ~or maintaining constant relative humiditias (Data o~ Solomon, 1952) % 'ileight in gm. Weight of water RelatiV'El o~ H2S04 per in gm. per 100 gm. Ifumidity 100 gm. o~88olution solution. 100 0.0 100.00 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 20 57.76 42.24- 10 64.45 35.55 0 100.00 0.00 University of Ghana http://ugspace.ug.edu.gh - 20 - (d) l-E1'liODS OF PREPJJlING SPORB PRINTS FOR GERj.f.INATION T'FSTS Ev"nly scattered conidia for germination t0StS were obtained on glass slidGS am Van Tieghem glass covers by placing them about 1 cm beloH a diseased leaf held in a horizontal position. The leaf ',;"8.S then guntly tapped ~·ith a pencil to dislodge the conidia. (e) SPORE GERMINbTION TESTS i) The Slide Method Glass slides holding conidia W0re pl2.cvd on V-shapl3d glass roos in the humidity chamb0rs with tho spore-coated side facing upwards. ii) Jl1e Van Tieghem Cell Method This has been mentioned at page 15 under humidity chambers. iii) G0rmination on Hnst Leaf Leaf discs removed \-7ith No. 10 cork borer were uS8d for germi- nation tests on host leaf. The discs holdine the conidia were placed with the spore-coated side facing upwards on moist filter paper in plastic chamb8rs. The conidia wera germinated on leaf discs at 10~ R.H. only. (r) INC~TION Germinating spores v,'ere incubated in elbctrically-controlled incubators. Slides on the development of th~ conidiophores, forma~ tion of the conidia and release of the conidia were carried out at University of Ghana http://ugspace.ug.edu.gh - 21 room temper1lture - 26 .:t 2°C. SporG survival tests ,,,,r·· c·J.rri .. d out in a r "frigcir::.tor St:lt (c) ASSESSKEN'l' or CONIDIDII GERMINATION AND GERM roBE tENG'lR J.t th<. em of th~ d"sired incubation period, obsGrv,~tions w. . re made immediately ,'lnd counts taken. If observations could not be made at once, the slid"s or l",af' discs were IXlt into lab€:lldd p,-,tri disht:ls and storod in the; r"frigerator (2°C.) to stop gI'O'.rth (,m observntions made Inter. rl. conidium ',laS considered to have germinated if a g.;;rm tube was discernable. Assessment of conidium germination for each tru~tm",nt , :as bas ·A. on the percentage of conidia germinated and the m..:.an length of 30 measured germ 1l.lbcis. The percdntage germination was caJ.culntoo from 600 observed conidia randomly salect.;d. The "percontage germination" , :as calcu- lated from the forllUla:- ,100 x ""0. of germinahd conidia 600 The lengths of the germ tubes were measuroo, with a microm"ter eye-piece. .her" gtJrm tubes branched or whore a coni~lium produc t:ld more than onG germ tube, ",erm tub.' length was taken ::'.5 thu sum of the lengths of the main gvrm tube and those of th" brfl.llCh",s or the sum of the lengths of the individual germ tubes of a conidium. University of Ghana http://ugspace.ug.edu.gh 22 - In th" germination tHsts where obs"'rw~tions '.;:;:re made on the formation of appr3ssoria, p0rc~ntage of ~0rm tubws forming appresoria was c1l.lculated :£'rom the formula:- ~_~...of conidia forming appr"soria Total No. of g .)rminatc.o. conidia In the germination tests where obssrvations were ~e on the rate of shrivelling of germ tubes, percenta",6 of t.::rminated conidia \1ith shrivelled ,~(;rm tubes was calculated from fu J formula:- 100 x ,' 0. of E;(;;rmin.:tted conidia with shrivel~"d .germ tubes Total 1:0. of germinatsd conidia During s;,ort. longevity t~sts conidia a~)l" to germinate after desired storaGO periods m:re considered viable. (h) EXPEiUMEJIITAL PRECAU'rIONS 1. CornJr (1935) and Nour (1958), ~, rOClIl6 othors, havt:: noted that the gsrmim.tion ')f' conidia of powd"ry mildu.rs is v~ry irregular. In soms te::sts th, conidia showed a high P(;:l'C .mtage germin~\tion whereas in othar tests th(;re was very little g~rMination. To ensure reliable results e.nd to cu t down the irrIxlea held in atlllOspheree ,nth a relative humidity of 100 per c nt \iaS studied at temperature 1 , vela of 1.5, 20, 25, 30 and 35°C. This range: was adecpate to covor the possible rllnge of temperature th .: conidia will be subjected to in th , tropics. 'rho conidia wert' incubated at th" five different temperatur0s arxl at 1~', R.H. for the variou::: puriods as indicated in the tables of r .sults (Tables 3 to 7). TWl:llve slid", s with conidia were h01d Il.t each t "mperature. Three slid;s woro rOlJloved after 12,18,24 .:md 36 hours rtlspectively and the. p€:rc,,;ntage :,Jrmination was ostimatdd and the length of th e, germ tubes measured. University of Ghana http://ugspace.ug.edu.gh 26 'fhe r.;sults of these experim<.Jnts ::.r..; tabulated in TablE;s 3 to 7, and express~d graphically in ]'ig. 3. ThE; conidia germinated at all lE;vuls of temperature used, but germin"tion at 15 o.nd 30°C. was v!ory poor. less than 11 per cent of th<. conidia germinated at 15°C. in 36 hours and still fewer (4.6 per oent) ~erminated :>..t 35°C. '.·.i.thin th.:.. sam& period. Conidial germina- tion at 20°C. waS also low and there :fas only 22.0 per ct:nt germinn.- tion. l~t these temperatures (15, 20 and 3SoC.) perc c:ntage germina- tion ani germ tube growth shoVl. d closely parallel temperature r,) S~ lonse. The; optilllUm tempera tur,; ':13.S fo" nd to be 25()C. sup :.>Orting th;:; high(;st m(:an perc entage germinvtion of 60.1 in 36 hours. ,.1 though 30°C. '.;~ evidently sub-optimal (45.4 per c"nt germination in 36 hours), th ~" germ tubes wer '; slightly longd' ~t this temperature tha.n at 2SoC. The better grolrth of g.;,rm ·tuo .? s at 30°C. suggests tmt probably th ·. optilll1m temperature for SJ:.OJ.71ea lies b~tween 25 and 30°C. Jot all levels of temperatur<" exc i:lpt 1SoC., germination \;:1 5 such th:'.t almost all th.; conidia capable of gt::rminating had germinated ,lithin 12 hours, the shortest observation time used. Th,:;re was no inor .. ase in t:;ermination after 12 hours. It, hoy:ever, took conidia at 15°C. 24 hours to attain the maxilll1m purcc.:nt c,.ge germination. University of Ghana http://ugspace.ug.edu.gh - 27 - ':£ABLE 3: GarmiMtion of' Conidia of Phyllac~ corylea incubatod at 15°C. nnd at 100,% R.H. Time in &xperiment % lft,an Germ Tube Hours Num,er Germinatj')n Length in )l -~ 1 3.6 20.1 12 2 3.1 19.8 3 3.9 21.4 Mean 3.5 .- 20.4 ~ 1 2.7 22.0 18 2 4.8 20.1 3 2.5 21.1 --- Mean 3.0 21.1 -- ------. -,. ..... ! 1 16.2 28.0 24 2 9.0 25.3 3 5.1 21.0 Mean 10.1 24.8 ---........... 11.5 26.8 36 2 8.5 26.3 3 12.5 25.4 ----..........' .. Mean 10.8 26.2 University of Ghana http://ugspace.ug.edu.gh - 28 - Germination of: Conidia of: Phyllactinia. corylea incubated at 20°C. and at 10~- R.H. --_. . - - Time in ExpEJriment % Mean Germ Tube Hours Number Germination Length in .....- ---- )l 1 19.5 50.4 12 2 12.2 37.8 3 13.6 35.4 Mean 15.1 41.2 ~---. 1 17.6 44.1 18 2 22.4 31.1 3 12.9 36.4 Mean 17.6 37.2 .... , ... ~ .... ... ..-' 1 I 21.7 44.8 I 24 2 i 19.9 44.8 l I 3 18.-6 60.9 Mean 20.1 50.2 ............ - -1-._- 1 21.8 79.2 36 2 24.0 49.0 3 20.6 50.1 Mean 22.1 59.4 University of Ghana http://ugspace.ug.edu.gh - 2, - TABLE 5: Germination of ConidLt of Ph;yllactinia corylea incubated at 25°C. ani at 10~ R.B. Time in Experimunt ~o Mean G8rm Hours Number Germination Tube Length in 59.5 90.4 12 , 2 63. 6 80.6 i 3 58. 7 57.8 - -- Mean 60. 6 76.3 1 58. 4 90.0 18 2 56. 4 86.2 3 5-6.1 56.8 Mean l_.:!:..O 77.7 --, ...... I 1 ! 60.0 111.0 24 2 56.7 78.8 3 60.0 89.0 - Mean .- 58.9-- 92.9 ............. .. 1 59.8 86.9 36 2 59.1 91.5 I 3 61.5 93.3 Mean 60.1 90.6 University of Ghana http://ugspace.ug.edu.gh - 30 - TABLE 6: Germination or Conidia of Phyllactinia corylea incubated at 30°C. and. at 10(J}c R.H. --- - - -.. 11can Germ Tube Time in Experiment ~ Hours Number G-erminn. tion I,cngth in )l - 1 4-9 .8 83.7 12 2 42.~ 9 I 69.0 3 50.. ~ .. ~._. 61.6 Mean 4-7. 7 ! 71.4- --_.+ .. _----- 1 4-0. 3 98.4- 18 2 4-5. 3 63.7 3 4-0. 9 68.6 Mean --_ 4-2. 2 76.9 ~ ... -- ........ l 1 39. 4 107.5 24- 2 48. 5 72.5 3 4-4-.9 111.3 ._-- Mean 4-4-.3 97.1 4-6.4- 108.2 36 2 42.9 128.8 3 46.9 119.4 Mc~n --- 45.4- 118.8 --.J University of Ghana http://ugspace.ug.edu.gh - 31 !rA.BLE 7: Germination of Conidia of Phyllactinia coryl<.?a incubated at 35°C. ani at 100% R.H. - Tims in Experiment % j,jean G(''Tm Tube Hours Number Germination Lenbth in f .-. -.. . - 1--' 1 4.1 16.1 12 2 3.7 30.0 3 4.3 26.3 Me(J.ll 4.0 2'. ... 1 ! 1 , 3.1 20.3 18 2 6.9 20.2 3 4.6 20.2 Mean 4.9 20.2 - ..... _. - 1 3.7 24.8 24 2 3.2 24.5 3 5.0 23.5 Mean I -1 4.0 rl --2-4.-3 -- 4.4 I 18.2 36 2 19.3 .3 ::: -j--- 19.2 Mean 4.6 18.9 .- --- University of Ghana h32t tp://ugspace.ug.edu.gh tOO -e--.-,2 HOUAI ......_ .-e_._ II HOURS __ e _ _ -e_ - ;Z.HOUAS ... J6HOU/u '0 '0 oz '0 ~ ~ ~ • ~O r 10 20 10 110 120 110 100 :' --, : '0 t --\: ( ' " I, r ",. ::/ 10 20 10,L--""---'-___~ _. .... 10 IS 20 :as 10 IS !"ECT 0' T!MPE.RATUAE ON TN. G.".,/IINATION U tt · . to..!. INCUBATED AT 100.,. A.H. University of Ghana http://ugspace.ug.edu.gh 33 - B. THE GER:;INATION OF CONIDIA OF P. CORYLEA AT VARIOUS RElATIVE HUMIDITIES The powdery mildews have attracted considerable attention by their ability to germinate at low humidities am extensive pertinent studies are reported in the literature (Cherewick, 19!;.4 j Naur, 1958; Clerk ani1l.yesu. . Offei, 1967; Yarwood, 1936; etc.). With the exception of very few species germination obtained ~t t~e lower IU1midities is rather low nnd the biological advantage of this habit is therefore limited. With species in which lOlVer IU1midities permit sufficient conidial germination, considerable infection units could be mobilisl1d under a wide range of atmosph~ric humidity conditions. The germination of conidia of P. corylea at vari~us relative humidities ani at 250 C., the optimum tempera:ture obtained in the previous experiment (See Table 5, page 29 ) Was investigated. This temperature ",1aB used in all subsequent experiments as tempera- ture of incubation, -:xcept " here otherwise stated. The conidia ",;ore incubated at varu..us rblativt: hUlUidities maintained. with sulphuric acid solutions (see page 35) as iniicated in the table of r 0sults (Table 8) for 24 hours, after ~;yhich (a) per. .. centage germination, (b) perc~ntage of germinated conidia which hq~ shrivelled, (c) p~rcentage of ungerminated conidia 'ihich he.d shri- velled (d) percentage of germ tubes bearing apprcssoria, were estimated and the l e ngths of germ tubes measured. University of Ghana http://ugspace.ug.edu.gh 3l. - It was obst3rved that to study germination of th.·; conidia in ?later on slid"s, SOffit; of t~ conidia floated whilst thtl r..;st remained submerged. To ~void cr0ating two different physiological contU tions, the conidia were incubated in sterile distillt:-d water in McCartney bottles (20 mls capacity) and thE> bottles were conti- nuously shaken for 24 hours on Griffin Flask ;haker at room tempera- ture of 27°C. Drops of Ifat"r with the conidia l1ere removc.-d at the end of the incubation period unto slides and percentage germination estimated. The results of th~sc experiments are tabulated in Table 8 and expressed graphically in Fig. 4 (Page 42) and also illustrated in Plates 5 - 13. The data in Table 8 showed that conidia of p. coryle:a germina- ted at all the humidity levels used. Germination .ms surprisingly very uniform and very high at any humidity from zero to 1 O~" R.H. The mean percentage gemination recorded for 1 OOJ~ R.H. ,Tas 41.0 and that for o:~ R.H. was 52.3. Germination was superior at any humidity level 1Xl that in liquid water, where a mean percentage B'8rmination of only 10.0 occurred. Unlike the very uniform percentage germination obtain...:d, the effect of different humidities was distinctly reflecbd in 6<;)rm tube growth. The germ tube length increased with riBe in relative humidity. Conidia germinating at Qro R.H. produced tho shortest germ tubes (mean University of Ghana http://ugspace.ug.edu.gh - 35 - TABToE 8: Effect of Relative Humidity on the germinati("m of Conidia of p. corylea incubated at 25°C. for 24 hours. (Relative Humidity maintained with Sulphuric acid solutions). ~- ---~ %S hri- 70 3hri- ~o of Mean velled vel led germ rubes germ % Experi- %G -ermi- germi- ung c.rwi- with rube Relative ment nation nated r.at rId appre- length Humidity Number conidia conidi a ssoria in)l -- .-. 1 21.6 : 0 1 0 28.5 64 0 8 1 ;i/ater 2 i 0 0 28.6 41.7 __ ~_ -Gl i 0 \ 3.8 0 15.6 43.) I I .. !I -~ ~ Maan ~~o.o i 0 L 24.2 49.8 ------f-- -._. ._ --1 1 35.5 1.8 2.5 14.4 88.6 100 2 46.3 I, 0.8 0.7 35.5 100.2 3 41.3 0.8 1.3 17.9 100.5 -- Mean 41.(0 1 .1 1.5 22.6 99.1 -.. .... - - -.. -- t- .---f-._ .. - ~-- 1 46.0 4.2 2.9 66.0 61.6 i 90 2 1 49 .5 4.0 I 59.3 53.9 I i 3 36.9 1.7 ! 51.6 54.3 __ :---_.._ -+-----+-.-- - _. ~ .. --t-I - ___i ----~ xa~ _---l~_44_.1_ _..: __3_ .4_._.! ___ .~·.~._I 59.v 56.6 University of Ghana http://ugspace.ug.edu.gh - 36 - Table B continued !:ff'ect of' :'telative Humidity on tht. ~;"rmin").tion of' conidia of' P. corylea incubated at 25°C. f'm' 24 hours. (Relative Humidity maintained with Sulphuric acid solutions). ---...... - .. ..- ~. - %S hri- I' ) Shri- ~o of' Mean % Experi- %G ermi- velled velled germ tubes germ Relative ment nation germi- ungermi- with appr&- tube Humidity dUJOOer nated nated ssoria length conidia conidia in}l ---~ - ~ - 38.B ! I 1 22.3 12.1 , i 51.2 48.6 BO 2 54.2 22.2 9.6 61.7 35.0 3 45.4 13.3 7.5 65.4 53.6 -. Mean 46.1 19.2 9.7 59.4 45.7 -- 1 41.B 15.0 10.7 62.6 43.8 70 2 53.B 28.9 19.2 53.B I 56.0 3 41.6 B.5 2.4 53.2 50.1 1-- Mean 45.7 17.5 10.B 56.5 50.0 - 1 43.6 19.5 18.0 66.8 40.2 60 2 48.3 20.3 23.1 49.6 43.4 3 46.1 10.3 4.8 57.5 42.7 t--- Mean 46.0 16.7 15.3 58.0 42.1 - University of Ghana http://ugspace.ug.edu.gh - 37 - Table 8 contiruail Et'f.;;ct of Relative Humidity on the germination of conidia of p. corylea incubated at 25°C. for 24 hours. (Relative HUmidity maintained with Sulphuric acid solutions). r~ ;:ihri- ~~ Shri- % of Mean ~ , Experi- %G ermi- veIled veIled ~rI!l iubes germ I" Re lative ment nation germi. . ungermi- with appre- tube Hu midity Number nated nated ssoria length ! conidia conidia in? -- .. ~ --- 1 44.6 I 13.0 4.6 61.0 32.5 I 50 2 54.4 7.7 3.8 58.7 39.9 3 49.5 4_.4_ . 2.2 52.0 35.7 . Mean 49.5 8.4 3.5 57.2 36.0 - ........ .. --- 1 48.2 12.2 9.7 64.5 32.5 40 2 50.5 14.5 2.8 47.9 43.4 3 53.0 23.0 19.3 54.5 28.3 - I -Mean 50.6 16.6 10.6 · 55.6 34.7 .. _.,....- ..... ..... ...--- _. --------- 1 51.3 25.3 20.0 63.3 24.1 30 2 52.2 26.3 23.5 54.3 29.7 3 47.7 20.3 20.4 .,--_ 54.0 31.8 .. Mean 50.4 24.0 21.3 57.2 28.5 ,... .. ,,- , University of Ghana http://ugspace.ug.edu.gh - 38 - Table 8 continued Effect of Relative Humidity on the ~~rmination of conidi~ of P. corylea incubated at 25°C. for 24 hours. (Relative Humidity maintained with Sulphuric acid solutions). --~I· .--_... --, ~vSeSllhredi-::II-%vOe'Sll'hr'e-df':l' germ~O Mean tu°bfes % E.xperi-. ~~ Germi- germ Relative ment I nation germi- ungerm:i:- with appre- tube Humidity Number : nated nated ssoria I length ___ . ___ -__ ~~' _____ +-c-o-n-id-ia...-Coniclia I I in ,u i j' 48.1 38.1 22 .8 72.3 19.9 20 2 48.0 16.6 12 .9 23.9 40.9 .1 57.9 28.4 ___- +-M_e_a_:_-+_ _:_ :_::_-I-_::_:_: :j: ::~...~3 - 51.4 29.7 .8 , 65.5 31.5 I 10 2 45 0 9 5 2 .1 ! 37.0 52.8 I \ I ! ~ 1I 3 45.5 I 14.7 6 .5 ,i 52.4 34.3 ! r--- - ! 44.5 21.2 11 .5 51.6 39.5 Mean - -- .. - 1 56.1 47.7 45 .6 39.5 24.9 0 2 60.6 17.0 20 .6 59.3 26.3 3 40.2 22.5 27 .1 44.0 30.4 - Mean 52.3 29.1 31 .1 47.6 27.2 - University of Ghana http://ugspace.ug.edu.gh 3? germ tube length of 27.2p) whilst the longL'st 1;;wrm t)'.lbE:ls (mean germ tube length of 99.1 p) W0re found in oonidia inoubated at 10q% R.H. Manners ,,-nd Hossain (1963) working on ~a~~ stated that at 985'f R.H. and 10~& R.H. good groVith ooourred at silitable tempera- tures and appr<::ssoria were produced abundantly. The pre::..ent work did not portray any rel".tionship between humidity level ani degree of formation of the appressoria. It was however found that the number of appressoria formed was oonsistently lower in Hater and at 10O}b R.H. than the lower humidities of 0 - 9ajo R.ri. The poor deve- lopment of appressoria in water and at 100% R.H. (where condensed water often appeared) may be due to the liquid \fater which seemed to prevent an establishment of a ::'irm attaohment by the; g~rm tube unto the glass surface. The development of a@ressori:-. in germ tubes of conidia incubated and shaken in water indioate that contact with a solid substratum is not a ~ .9Y.!!: .!!Q!! for appressol'ial formation. Low humidity always caused oonsiderable shrivelling of both germinated and unbGrlllinated conidia of powdery mildews and this desiccation action is more pronounced at higher temperatures (Yarwood,1936). The results of tests of the relation of temperature to desiccation of clover mildew and mustard mildew shom~d that the percentage of shrivelled conidia in the mounts over snlphurio acid was :orogressively Gre:··. t, ;r as the temperature increased. Yarwood's University of Ghana http://ugspace.ug.edu.gh - 4-0 - observation that volume changes of the non-5~rminated conidia was about too same as thn.t of the germinated conidia makes it difficult to infer whl;)th,r he associated the decrease in volume with the germination process or as an effect of low lrumidi ty. In 1952, Yarwood worldng again on E. graminis came to some positive conclusions. He stated that germinating conidia lost more water than non-germinating conidia and that while both germinating am non-germinating conidia shrank in a dry atmosphere, the germina- ting spores shrank: at a faster rate. Tlus, the germinating spores decreased in volume at about three timas th[;J rate of non-germinating spores. Brodie and Neuft:ld (194-2) working with E. polygoni failed 11:1 observe any changes in volume of the conidia germinating under laboratory conditions at a relative humidityof' from 65- - 80%. They positively concluded that the process of gerlllinatiun of conidia of E. polnjoni undtcJr the conditions of their studies was not acco&- panied by any changes in volume. Ayesu-Offei (1966) observed that both germinated and ungermi- nated conidia of I,. taurica shrivelled when incubated at various relative humidities. He however noticed that, unl:ilce observations of Yarwood (1952) germinated and ungerminated conidia of Leveillula ~ shrivelled to tiE same extent and did so (~t the same rate. University of Ghana http://ugspace.ug.edu.gh - 41 In the present studies germi.nat"d conidia and ungerminated ones shrivelled to the same degree. The perc"ntat;e of conidia which Shrivelled incrvased with decrease in humidity until a. level of 30% R.H. A further fall :in humidity failed to .:l.ffect any further rise in quantity of shrunken conidia. University of Ghana http://ugspace.ug.edu.gh - 42 - 100 I eo i 1 60 z .-.-_........ .---.......... 0 -. / ~ 40 .- ! 2 a: III 20 "t- o 140 120 ~ ! 100 .x..: ~ 10 '~" CD .:'.;".) 60 2 It ~ 40 z c '~" 20 ·/0 RELATIVE HUMIOITY FIG.. . EFFECT OF RELATIVE HUMIDITY ON GERMINATION -OF ' CONIDIA OF P.CORYLEA INCUBATED AT 2S·C. FOB 24 HOURS. University of Ghana http://ugspace.ug.edu.gh ~ -~ ~ "It ,I~ "" ', ".. \. I -.~ \ I / I PLATE 5: Photomicrograph of' conidia of P. corvlea germinating at 0.0% R.H. and at 25°C. af'ter 24. hours incubation. Note: Shrivelled germ tubes and collapsed conidia. X 110 University of Ghana http://ugspace.ug.edu.gh - 44 - PUTE 6: Photolllicrograph of conidia or P. coplea germinatiDg at 12.0% R.H. and at 2S0C. af'ter 24 hours incubation. Note: Moat germ tubes have shrivelled. X 110 University of Ghana http://ugspace.ug.edu.gh .~- PLATE 7: Photomicrograph o~ conidia o~ P. coryl ea e;erninating at 33.2% R.R. ani at 25°C. af'ter 24 hour8 incubation. Note: )(08t germ tubes bne shrivelled. I 110 University of Ghana http://ugspace.ug.edu.gh - ,.,6 - PLATE B: Photomiorograph of conidia of P. oorvlea gerlllinating at 5'.~ R.H. and at 25°C. after 24 hours incuba. tion. Note: ShriTelling of just a few gerlll. tubes. X 110 University of Ghana http://ugspace.ug.edu.gh - ,.,7 - PLATE 9: Photollicrograph of conidia of P. cOrylea germinating at 75.9.' R.H. am at 25°C. after 2Jt. hours incubation. Note: Most gera tube. were turgid. X 110 University of Ghana http://ugspace.ug.edu.gh - 4B - .1 - PLATE 10s Photo.icrograph of conidia of P. corvlea germinating at 80.3~R.H. and at 25°C. after ~ hours incubation. Note: Most germ tube8~e turgid. X 110 University of Ghana http://ugspace.ug.edu.gh - 4.9 - PLA'l'l!: 11: Photomiorograph ot conidia of P. cor;rlea gera1na~ at 92."" R.ff. and at 2SoC. after 24 hours incubation. Note: Turgid and. long gerll tubes. 1: 110 University of Ghana http://ugspace.ug.edu.gh - 50 - PLAn 12: PhotoaicroUapb o~ conidie. o~ p. carrIea gerainating at 96.~ R.H. and at 25°C. after 24 hour8 iDCuba tion. Note: Turgid am long gera tubes. I 110 University of Ghana http://ugspace.ug.edu.gh - 51 - PLATE 13: Photomicrograph of conidja of P, corylea germinating at 10~ R.H, ani. at 2SoC. after 24 hours incubation. Note: Long and turgid gera tubes. X 110 University of Ghana http://ugspace.ug.edu.gh 52 C. l!URTHER EXPERI~S ON THE GERFINATION OF COlHDIA OF P. CORYLEA AT VARIOU~ REIATIVE HUl®ITJES (a) GERUNA.TION OF THE COJ'HDJA AT VARIOTY', REIAT:rvE !llL1.1J])I'fIES WITH SA.'lURA.TW J',.')UEOUS MoLT SOLUTIONS It was observed in the previous experiment that germination was very uniform ov(;;r the: range of' humidity usad (0 - 1 O~ R.H.) (Table 8). It was necessary to f'ind whether this was tho true behaviour of' conidia of' f. corylea. ~ermination over the same range of' humidity ;Ias theref'ore examined again, this time using saturated aqueous salt solutions to maintain th~ relative humidities according to the data of' ' ,exler and Hasegawa (19.54) (See page 18). The speci:f'ic hVHls of' humidity employed are shown in the tables of' results (Tables 9 ~'.n:l 10) and the con:idia incubated f'o1' 24 hours. As a further con:f'irmation two levels of' temperature, 20 and 250C. were used. P. rc0ntage germination \ms estimated, B~rm tube length measured and perc0ntage of' germ tubes showing apprbssoria assessed. These are presented in Tables 9 and 10, and in Fig. 5. The data in Tables 9 and 10 showed that conidia of' P. corylea germina ted we 11 and very uni:f'orinly at all the humidities (0 _ 100li R.H.) maintained with saturated aqueous salt solutions incubated at 0 both temperatures of 20 and 25 C. ~ermination at 10~, R.H. at 250C. was 56.9 per cent and th~t at Q% R.H. at the same temperature Was University of Ghana http://ugspace.ug.edu.gh 53 - 50.5 per oent. At 20°C., 41.2 per cent of the conidia germinated at 10(1, R.H. whilst 39.9 per cent d:id so at oy~ R.H. Germination was lower at 20°C. than at 25°C. at each corresponding relative humidity. The data showed that the high~r the humidity the longer the germ tube length. Thus, at 25°C. 10~JR.H. showed mean germ tube length of 112.6)l whilst at CJ% R.H. the mean germ ulbe length waS only 17.3 /u. At 20°C. the mean germ tube length at 100'}'0 R.H. was 46.7)l and that at 0;: R.H. was 19.41. Appressorium formation again followed no definite pattern, indicating that relative humidity has no influence on apprtlssorium development. The data in these experiments conformed to the behavi~r of the con:idia of p. corylea found in the previous experiment. University of Ghana http://ugspace.ug.edu.gh - 54 - TABLE 9: KITed of Relative Humidity 011 'i;he" Germination of Conidia of P. corylea incubated at 20°C. for 24 hours. (Relati vc humidity maintained with saturatod a.~eous salt lIolutions). r- Saturated yo G€rm Mean germ % a~eous Experi- r~ Germi- tube ';lith tube length Rclative salt zolu- ment nation appro s S('ria. in )l fumiditY tion usod Number - ,..- 1 27.4 38.1 29.1 2 41.4 8.4 44..1 100 WATER 3 54.9 21.1 66.9 Mean 41.2 22.5 4-6.7 . ---~ ---- 1 30.5 48.8 24.2 2 4-7.2 17.7 4-2.3 ~S04- 3 53.9 23.4- 49.0 Mean 43.9 30.0 38.5 .- t----- -- ~- .. ~ . 1 29.6 37.6 39.2 2 4-2.1 23.4- 44..1 93.2 KN0 3 3 53.9 26.1 52.2 Mean 4-1.9 29.0 4-5.2 .-- -,,---..... University of Ghana http://ugspace.ug.edu.gh - 55 - Table 9 continued .Eft'ect of kelative Humidity on the Germination of Conidia of P. corylea incubated at 20~C. for 24 hours. (Relati ve Humidity mainbint. \ ~ \ \ ~ a: 40 \ 20 UJ ()..-o..~O C) 20 10 0 100 60 60 40 20 0 0/0 RELATIVE HUMIDITY FIG . 7. EFFECT OF RELATIVE HUMIDITY ON THE GERMINATION OF CONIDIA OF P. CORYLEA INCUBATED AT 27:t 10C. FOR 24 HOURS. University of Ghana http://ugspace.ug.edu.gh 69 D. EFFECT OF FLUCTUATING ATMOSPHERIC HUHIDITIES ON THE GIilRmNATION OF C01'lIDIA OF P. CORYLEA. AT THE TEMPERATURE OF THE Am The previous experiments on germination of the conidia have been carried out under constant humidity conditions. In nature, the spores are invariably subjected to fluctuating humidities. It is probable that where the changes in rumidity are abrupt, stresses may be impcsed on the membranes of the spores especially at the period of onset of germination when the spore has lost protection of the quies- cent state, which may lead to disfunction of the conidia. The germi- nation of conidia of P. corylea exposed to atmospheric hum;dity on glass slides was examined. Dry slides bearing conidia on the upper surface were exposed to the atmospheric humidity on an open veraIliah for 24 hours. The conidia were collected and incubated at 3 p.m. of the day ~f the experiment and the percentage germination estimated and germ tube length measured at 3 p.m. on tba following day. The data for the daily and hourly atmospheric humidity of Legon, (Fig. 8) were compiled from the weather charts o~ Department of Geography, Univer- sity of Ghana, Legon. This department is situated south-east and 200 yards away from the Botany Department. University of Ghana http://ugspace.ug.edu.gh 70 - INITIAL TIME OF INCUBATION ~IOO a __ •_ ______ •_ _ O -F- -C__O_N__I_D eIA--:- --_\ 3 ·00 PM 14: 2.:/6_8 i ~ 80 .' •~ \ .I ~ j \ .... -./ 3 60 - ."j .... Ill: "! 40~~~~Li~Li~Li~Ll~LL~LL~LLJ-LJ 2PM 6PM IOPM 2AM bAM 14:2:68 15:2:68 INITIAL TIME OF INCUBATION ~ 100 a .-.-..... --.-.O. F-. .C..O...N...I.D..I.A...:. ..... 3-00 PM. 19:2:68 i ~~ \ . / ~ 80 .... . / ~ \ W'W'• ~ ~~. ...... ~ 60 u./ Ill: "! 40~-L~~~Li~Li~~-L~~~LL~Li-L~~ 2PM 6PM 10 PM 2AM 6AM lOAM 2PM 19:2 :68 20:2:66 100 INITIAL TIME OF INCUBATION OF CONIDIA : ],00 PM. 20:2 :68 .>..-. j ................... _-----.\ ~ " 80 ~ :r _i \ ~~/ .-., ...... ,., 4 "2 PM 20:2:68 TIME IN HOURS FIG . 8 . RELATIVE HUMIDITY OF THE ATMOSPHERE A5 RECORDED BY A THERMOHYGROGRAPH DURING GERMINATION OF CONI[\IA OF P .COPYLEA UNDER ATMOSPHERIC HUMIDITIES. University of Ghana http://ugspace.ug.edu.gh 71 FroID the data of the results (Table 12) (:t>a.ge 73 ) it would be seen that ~rlDination was high and uniform for the three days. On the 14th February 1968, germination was 63.9 per cent though the humidity rose from Ei4.c% R.H. at 3.00 p.m. to 86.8~o R.H. at 6.00 p.m. the oritical period for germination. On the 19th February 1968, germination was 80.4 per cent when the atmospheric humidity shifted from 84.% R.H. at 3.00 p.m. to 8~o R.H. at 6.00 p.m., ani on the 20th Febru ary 1968, germina tion was 74.9 per cent. This happened under atmospherio humidity which changed from 69.~o at 3.00 p.m. to 88.1% at 6.00 p. m. It was inter~ sting to note that wher e th~ change in humidity was sli~t in the latent period, (84.0 to 89.~~ R.H.). germination was better (80.4%) than when there was a considerable change. (For example, 63.9 per cent of the conidia germinated when the humidity shifted from 64.0 to 86.8% R.H. The latent period of germination of P. corylea as will be shown later (see page 83 ) was between 2 - .3 hours. The conidia of P. corylea therefore germinated very well within this period when the atmospheric humidity changed rather sharply between 3.00 p.m. and 6.00 p.m. on the days of the experiment. Extensive studies have been carried out to determine the ability of conidia of various members of ~8iphaceae to germinate under natural atmospheric humidities. A comprehensive list of such record University of Ghana http://ugspace.ug.edu.gh - 72 - on Erysiphe graminis extracted f'rom a similar <-ompilation by Brodie (1945) on some powdery mildews is presented. in Table 13. By compari son, conidia of' P. corylea belong to the category of' those powdery mildews oapable of' germinating very well unier atnDspheric humidities. University of Ghana http://ugspace.ug.edu.gh - 73 - TABLE 12: Germination of conidia of P. corylea on dry slides expesed to natural Atmospheric Humidities at atmospheric temperature for 24 hours. Experi- Initial time Average Mean ment Time of % Atmosphe- germ tube of Germi- Number Incubation Observation ric Tem- nation length in perature )l 1 3.00 p.m. 3.00 p.m. 29.0oC. 63.9 87.2 14/2/68 15/2/68 2 3.00 p.m. 3.00 p.m. 28.3OC. 80.4 97.0 19/2/68 20/2/68 3 3.00 p.m. 3.00 p.m. 28.8°C. 74.9 90.1 20/2/68 21/2/68 University of Ghana http://ugspace.ug.edu.gh - 74 - nBLB 13: Reported data on germination of conidia of Erysiphe e;raminis exposed to a tmospheric humidities. Tempera. . Atmosphe- % Host species ture °e. rie Germi- Author Date fumidity naticn ~ vulgare 23 - 20 Brodie 1945 " " 23 - 22 Brodie 1945 " " 23 - 19 Brodie 1945 " " 21 40 12 Brodie 1945 • • 21 40 33 Brodie 1945 .. " 20 50 14 Brodie 194-5 " " 20 50 22 Brodie 1945 I tt " 20 46 29 Brodie 1945 " " 18 - 9 Cherewiek 1944 ~ aestiVUII 22 58 35 Brodie 194-5 " " 22 43 30 Brodie 1945 " It. 22 41 40 Brodie 1945 " " I 18 - 40 Cherewick 1944-- -- University of Ghana http://ugspace.ug.edu.gh - 75 - Table 13 continued Repcrted data on germination of conidia of Erysiphe graminis exposed to atmospheric humidities. a.rempera- Atmosp~e- % H Host species ture °C. rie Germi- Author Date Humidity nation ~~ 18 - 16 Cherewick 1944- !!Q! 2ratensis 22 - 15 Brodie and 1942 Neufeld Agropyron repens 25 - 18 Brodie 1945 I " " 23 i - 30 Brodie 1945 I ! I Agropyron !£. 18 I - ! 20 Cherewick 194.4- Delphinium ~. 22 - 30 Brodie and 1942 Neufeld Phaseolus wlgaris - 50 25 Brodie 1945 Brass10a oleracea - - 35 Clayton 1942 ~!£. 24 61 5 ! Brodie 1945 ~sp. 22 65 16 Brodie 1945 - - _o_e_n_o_t_h e_r _a_ _. _ ______ ~ _____ ~ _____ ~~_y_t_ o_ n __~ __1 _9_4_2_ University of Ghana http://ugspace.ug.edu.gh 76 - E. EFFECT OF VARIOUS RELATIVE HUMIDITlES ON THE RATE OF GERMINATION OF CONODIA. OF P. CORYLFA It is now convincingly established that germination is equally goed over a very wide lUlmidity range (0 - 1 00% R.H.). The length of germ tubes of the conidia was however not uniform am using this value as a criterion it could be suggested that the more favourable lUlmidities (higher humidities) would support longer germ tubes than the less favourable ones (lower humidities). Another useful criterion which can be used is to examine the rate of germination of the conidia at the various relative humidities. More favourable lUlmidities will encourage faster rate of germination and ~~. The following acccunt reports of tiniings of' this investigation. The conidia were incubated at different humidity levels as shown in Table 14., maintained with saturated aqueous salt solutions at 25°C. for only 12 hours, since experiment in chapter 1 iniicated that the conidia gave maximum germination for eaeh humidity level within that period. Four sImes bearing spores were plaoed in each humidi ty chamber. . Two were withdrawn after 6 hours ani the remain- ing two after 12 hours and (a) peroentage germination estimated, (b) percentage of germ tubes with appressoria assessed, and (c) germ tube length measured, for eaoh period of incubation (6 hours and 12 hours). University of Ghana http://ugspace.ug.edu.gh n - The results are shown in Table 14 and illustrated graphically in Fig. 9. The results showed that at 6 hours, the earliest time o~ observation, germination was approximately un~orm at all relative humidities, and similar to values obtained after 12 hours incubation. It seems that for a oritioal estimation of rate of germination of these oonidia, shorter intervals are neoes sary. University of Ghana http://ugspace.ug.edu.gh - 78 - TABLE 14: Effect of Relative Humidity on the rate of germination of oonidia of P. corylea incubated at 25°C for 12 hours. INCUBATION PERIOD IN HOURS % Expert:.:: 6 12 Relative ment Germ Germ Mean lfu midi ty Number % Gerlhi- tubew1th ~~~~ % Germi- fubewith germ appre- gth in appre- length nation ssoria nation )l ssoria inp 1 59.0 12.5 85.4- 57.2 23.1 150.9 2 68.2 15.4- 90.7 71.6 11.3 144.6 100 3 80.2 9.4- 86.8 79.4- 9.2 147.4 Mean 69.1 12.4- 87.6 69.4- 14.5 147.6 1 55.9 10.9 94.9 65.7 19.1 120.0 2 70.2 13.5 85.7 75.2 7.7 168.0 96.9 3 91.1 21.7 77.4- 90.5 29.0 109.6 Mean 72.4 15.4 86.0 77.1 18.6 132.6 1 58.5 8.5 106.4 53.8 21.0 113.1 2 73.2 16.2 85.0 67.9 7.6 116.6 92.0 3 86.6 20.7 75.9 88.5 22.0 117.2 - Mean 72.8 15.1 89.1 70.1 16.9 115.6 University of Ghana http://ugspace.ug.edu.gh - 79 - Tabla 14 continued Bffect of T:elative Humidity on the rata of germination of oonidia of P. corylea incubated at 25°C. for 12 hours. INCUBA TION PERIOD IN HOURS % Experi- G 12 Relative ment Number % Germ Mean germ Germ Mean Humidity Germi- tute with t.ilie len-- Ger%IlU' - tul:e with germ nation appre- gth in nation appre- length ssoria p. sscria in )l 1 .54.2 11.0 73.5 62.2 18.1 93.5 2 75.2 3.6 I 92.7 76.3, 17.4 120.0 80.3 3 85.6 21.4- 51.8 91.5 18.4 96.6 Mean 71.7 12.0 72.7 76.7 18.0 103.4- 1 56.6 14-.5 67.2 53.3 15.7 85.7 2 63.9 7.6 72.8 66.0 13.8 79.4- 75.8 3 84-.8 15.3 74-.6 85.1 8.8 91.0 - Mean 68.4- 12.5 71.5 68.1 12.8 85.4- 1 58.4- 10.3 68.6 63.3 12.6 81.2 2 72.7 16.0 63.3 72.7 13.0 70.3 53.8 I 3 I 85.3 13.5 61.3 85.6 19.0 75.6 Mean 72.1 13.3 64-.4- 73.9 14-.9 75.7 University of Ghana http://ugspace.ug.edu.gh - 80 - Table 14 continued E!'t'ect of Relative Humidity on the rate of germin.?tion of conidia of P. corylea incubated at 25°C. for-12 hours. INCUBATION ~IOD ill HOURS ~ Experi- 6 12 Relative ment Humidi'bJ Nu:;)ber % Germ illEan germ ~ Germ ]'.[D Germi- ~wlth Itube len- GerL'li- tube with G3rm nation apprl3- 16t~ natior. appre- length in ssoria ssoria in)l 1 58.8 12.6 62.6 57.1~ 13.6 63.4 2 63.7 8.1 I 71.1 72.0 9.0 75.6 33.2 3 83.3 9.2 60.9 37.3 10.7 67.6 Mean 68.6 10.e 64.9 72.2 11.1 68.9 1 57.3 5.7 54 . 3 .5L: .• 2 4.3 56.7 2 67.1 9.8 i 50.8 65.8 2.5 59.8 12.0 3 79.2 6. "' 51.5 I 83.5 6.5 52.8 Mean 67.9 7 ,4 52.2 1 67.8 4.4 56.4 1 54.8 8.2 52.9 53.3 9.3 53.2 56.6 14.2 1~7 .2 65.2 12.2 53.6 ~: 86.7 7.0 52.2 86.1 14.7 1·~3.3 - ----66.0 9.8 50.8 63.2 12.1 50.2 University of Ghana http://ugspace.ug.edu.gh ISO 100 LEGEND 1lZiJ0IoGERMINATION CJGERM TUBE LENGTH 90 130 .0 110 f) 7. r- 70 17 V. 'I r"l ll V ~ / V V V / 60 Vv / /11II IIVV II 90 II 1~I 'I) IIII ~ II V 50 fI "I~ 70 II II II ') II fI ~r ~ 6 40 ~ II j ~ HOURS. V II / / V II / I ~ 1I V Vv / 50 30 ') 20 VV ~ II II 1II ~1/ II I fI V 1/ II 30 fI 1/ ') II II fI II II 10 1,1 ~ 1/ ~ II tI fI O~ LL~ ~~ ~ 10 ~~V WL~ LL~ ~~ ~~~~~ 100 ISO r- 90 130 o 110 o o 90 o 12 70 HOURS SO 30 10 100 96·9 92-0 80·3 75·8 53·e 33 ·2 12 0 °'0 RELAT IVE HUMI DITY FIG. 9 . EFFECT OF RELATIVE HUMIDITY ON THE RATE OF GERMINATION OF CONIDIA OF P.CORYLEA AT 2SoC . University of Ghana http://ugspace.ug.edu.gh 82 - F. roRTHER EXPERIMENTS ON 'lHE EFFECT OF VARIOUS IillMIDITIES ON THE RATE OF GERIliNATION OF CONIDIA OF E. CORYLTiA Most studies on spore germination employ as cri ter:iDn the final germination of a population to determine the effect of the environmental factors on germination. Another major criterion often used is the latent period of germina.tion defined as the time requ:ired for eit~r germination to begin or the percentage germination to reach some specified lew value (Bonner, 1~8; Groom and Panisset, 1933; Tomkins, 1932) 0 In the previous experiment (page 77 ) the conidia at every humidity level tested showed the possible maximum germination after 6 hours. Percentage ge~mination Was besidds uniform over the entire humidity range. The search for the optillllm relative humidity for germ ina tion of conidia of P. corylea could be exterrled further to either examining the rate of germination within the 0 _ 6 hours interval after inCUbation or finding the latent period of germination of the conidia. The dxperiment below was designed to investigate both. The conidia were incubated in the usual manner at the different humidity levels shown in Table 15 at 25°C. for 6 hours. Humidities were maintained with saturated aqueous salt solutions. Twelve slides bearing spores were placed in each humidity ohamber. Two were with- drawn at hourly intervals for a total period of 6 hours and percentage University of Ghana http://ugspace.ug.edu.gh 83 - germination estimated. The results are pres6nted in Table 15 and Fig. 10. The latent period of germination defined as "the period or time required by 5oy,; of total germinable conidia to produce germ tubes" was then calculated from the graph in Fig. 10. The eff6cts of the various levels of humidi~ on the germinaw tion of the conidia were closely similar. The latent period of germination of the conidia of P. corylea at each humidity l~vel was between 2 and 3 hours after incubation. Besides, th6 conidia showed identical rates of germination over the entire humidity range. University of Ghana http://ugspace.ug.edu.gh - 84- - TABLE 15: Percentag~ of conidia of P. corylea able to germinate at various relative humidities at 25°C. % Experiment lNCuBlTIrn PERIOD IN HOURS Relative ffilmidity Number 1 2 3 4 5 6 1 I 0.7 20.9 56.1 65.1 66.8 68.0 2 0.4 21.0 49.3 50.3 56.9 57.9 100 3 0.0 19.1 50.0 5B.9 66.8 69.8 Mean 0.4 20.3 51.8 58.1 63.5 65.2 ------- i I i I 0.9 13.2 23.2 l 43.B 50.B 51.0 2 I 0.2 20.3 45.5 47.9 55.5 49.2 96.9 i 3 0.0 21.1 53.6 61.0 68.0 70.3 -- --- Mean 0.4 1B.2 40.B 50.9 58.1 56.B 1 0.0 20.6 56.B 74.5 BO.8 80.9 2 0.0 92.0 I 1B.9 54.7 65.7 60.7 62.2 3 0.0 19.2 50.4 59.9 65.9 64.5 Mean 0.0 19.6 54.0 66.7 69.t 69.2 - University of Ghana http://ugspace.ug.edu.gh - 85 - Table 15 continued Perc .cntage of' conidia of P. corylea able t" germinate at various relative humidities at 25°C. % INCUBATION PERIOD IN HOHRS Relative Exp'Jrimen t 1---- Humidity Number 1 2 3 4 5 6 1 0.6 24.2 54.4 59.9 47.4 59.5 2 0.0 20.0 43.7 50.3 56.4 66.6 80.3 3 0.0 26.0 40.1 55.6 60.1 61.0 Mean 0.2 23.4 46.1 55.3 54.6 62.4 '._-_.1 - --- - ---- 1 0.0 26.8 30.2 52.7 68.0 76.9 2 0.6 20.9 34.8 49.3 52.9 50.1 75.8 3 0.0 17.3 59.1 71.9 72.5 75.2 - Mean 0.2 21.7 41.4 58.0 64.5 67.4 l 1 i 0.4 19.0 49.3 55.6 i 62.3 \ 63.3 I 2 0.0 18.6 \ 30.1 39.5 l 48.2 50.2 53.8 3 0.0 20.2 44.3 56.4 61.9 61.0 Mean 0.1 19.3 41.2 50.5 57.5 58.2 University of Ghana http://ugspace.ug.edu.gh - 86 - Table 15 continued Percentage o~ conidia o~ P. corylea able to germinate at various relative humidities at 25°C. % Experiment INCUBATION PERIOD IN HOURS Rel.?tive Humidity Number 1 2 3 4 5 6 1 0.9 20.4 42.2 50.0 50.1 52.5 2 0.0 12.7 39.9 40.5 45.5 - 46.1 33.2 3 0.0 19.9 51.9 58.9 60.4 60.2 ; Mean 0.3 17.7 ! 44.7 49~~ I 52.0 52.9 --- 1 0.6 24.3 57.3 65.9 69.0 69.5 2 0,0 6.1 24.9 43.1 54.7 56.9 12.0 3 0.0 26.5 49.1 65.0 -6_5 .5 64.0 .... _-I- Mean 0.2 19.0 43.0 58.0 63.1 63.5 ; 1 0.3 ! '18.5 4 31.8 4507 51.3 51.5 1 2 0.1 27.6 I 57.2 66.5 70.1 69.5 0.0 1 I 3 0.0 8.3 39.8 53.1 60.2 59.3 ~----- Mean 0.1 18.1 42.9 55.1 60.5 60.1 -. University of Ghana http://ugspace.ug.edu.gh 87 - ____________ --------,100 0z jso .r- :",i lOO°JoRH. f ~. ~O CI Z 'OO Q % ~.r-i 96 '9 R.H. IL ___- ""'~e::::....----'--.----------~O ~ '00 Z Q 92'0 % RH · ~ 50.r-ai: L-_-A_ __~ __~ __________________1O ~ r----------------------,IOO ~ 80·3"10 RH. L-___~ __ __L=.._ ___ ______________l: o *i r-------------------------,IOO Z 75 ' 8 % RH . ----r- 150 .r-i L-----------------~--~-----------------o CI r--------------------,IOO ~ 53' 8 % R.H. ]~5 0 -0° ~~",' I L..- _.-~.::::...-..L.---------_O CI r-------------------------------,IOO ~ "'.~'" I ~ 1:° i~ I~-.-=---:--r-:----:--:- ------------,IOO Z "0·'_'" 1; *i Ir--------------------------,IOO Z 0°/0. R.H. j 50 .r- ~ · ~ Jo~ O 2 3 4 TIME OF INCUBATION IN HOURS FIG.IO THE EFFECT OF VARIOUS RELATIVE HUMIDITIES ON THE RATE OF GERMINATION OF CONIDIA OF BCORYLEA INCUBATED AT 2SDC . University of Ghana http://ugspace.ug.edu.gh 88 G. EFFECT OF HUMIDITY ON THE RATE OF GERM TUBE GROWTH 1b st fungi require a high humidity usually 95'f~ or more for growth, but a few, such as some grain deteriorating fungi, can tolerate humidities as low as 85% to 90% R.H. (Hawker, 1950; Lilly and Barnett, 1951; Cochrane, 1958). The previous experiments have sh0wn that: (a) the conidia of P. corylea germinate uniformly over the humidity range of C - 10<:% R.H., (b) that the rate 0:[' germination is uniform for this humidity range and (c) that the latent period of germination is approximately' similar. It.as impossible therefore to assess the comparative importance of these humidities in germination by these criteria. The only feature where variation has consistently been observed is the length of the germ tubes (Tables 8, 9, 10, 11 and 1~). It seems therefore that the rate of germ tube growth could be used to assess the rore favourable humidity for the growth of P. corylea. It will however be pointed out that this criterion is being used to embraoe both germination of the conidia and growth of the germ tubes more particularly. In an experiment set up to establish the influence of the humidities on growth of too germ tubes, percente.ge of germina- tion waS again uniform and occurred at the same time, and even germ tubes were quite similar in length at 6 hours (Table 15 and Fig. 10). It was after this stage did marked differences appear in the lengths University of Ghana http://ugspace.ug.edu.gh 89 ... of germ tubes, o~viously due to different rates of extensional growth. This dxperiment was again carried out by incubating eight glass slides bearing conidia of P. coryloa at 25°C. at each humidity (Table 16). Two slides werG removed at 6 hour-intervals and percen- tage germination waS estimated and measurements made of the lengths of 50 germ tubes for each tr"atllSnt. The entire experiment was extended over 24 hours. The mean lengths of the germ tubes are shown in Table 16 and are illustrated graphically in Fig. 11. The percentage of germina- tion was practically identical to those already recorded and so omitted from the table of results. The results (Table 16) imicate that f!!3rm tubes of conidia germinating at 0.0, 12.0 and 33.2% R.H. stopped growth after 6 hours. Those at 53.8 and 75.~; R.H. grew for 12 hours after which no further increase in length could be observed. Growth of the germ rubes at 10,,% h.H., however, showed a uniform growth rate, practically linear, over the entire period of 24 hours am it was evidently the most favourable humidity for germ tube growth. Germ tubes growing at 80.3,92.0 and 96.9,Po R.H. although showed extension thrOUthout the peric.:l of incubation, growth rate was var:iable. Two main periods were observed j in a first period of the 0 - 12 hour interval where growth rate was high and a period of very slow growth in the 12 - 24 hour interval, where the ra.te of growth was approximately a sixth University of Ghana http://ugspace.ug.edu.gh 90 - of' the f'or~r. The germ tub (;s c.t the lower rumidities did not only step groVith quite early during incubation but quickly b0came dry am shrivelled am probably dead. This raises Rn important question about the biological value of' the ability of' P. corylea conidia t:) germinate so profUsely at the lower humidities. If these conidia are not brought under the influence of' more favourable humidities after germination they are almost wasted instantly. University of Ghana http://ugspace.ug.edu.gh - 91 - T,\ELF. 16: Effect of various Relative Humidities on the rate of growth of germ tubes of P. corylea at 25°C. (Relative Humidity maintained with satur~,ted aqueous salt solutions). % Experi- G€rm tube length in ? at Relative ment indicated hours Humidity Number 6 12 18 24- 1 86.9 151.2 160.0 190.1 2 91.7 144.8 188.0 204.8 100 3 88.6 14.8.4- 180.2 199.0 -- Mean 89.1 148.1 176.1 198.0 - 1 95.6 119.1 136.1 140.2 2 86~0 167.2 163.4- 152.2 96.9 :5 76,6 108,9 147.4- 154.0 Mean 86.1 131.7 149.0 148.8 1 106.3 112.3 105.3 111.9 2 84.9 116.8 112.1 123.2 92.0 I l 3 75.8 116.2 126.0 145.2 Mean 89.0 115.1 114.5 126.8 University of Ghana http://ugspace.ug.edu.gh - 92 - Table 16 continued Effect of various Relative Humidities on the;, rate of growth of germ tubes of P. corylea at 25°C. (Relative Humidity maintained with saturated aqueous salt solutions). -- % EXPer~~-1 Germ tube l e ngth in f1 at ment indic ated hours Relative Humidity Nu mber 6 12 18 24 -- 1 73.4 93.4 106.0 116.5 2 91.9 119.9 121.4 122.7 80.3 3 52.6 95.6 99.4 97,0 Mean 72.6 103.0 108.9 112.1 .- ._-~.--- 1 66.9 85.7 79.5 84.4 2 72.8 79.3 76.3 81.2 75.8 3 75.6 90.9 92.8 94..2 -.. ----""--- Mean 71.8 85.3 82.9 86.6 5JT 68.6 81.3 79.1 80.1 64.3 70.2 76.3 63.4 i 3 60.3 76.2 86.4 87.8 _._._--- ;,jean 64.4 75.9 80.6 77.1 --_.,. .... . _- k .l ..._ __ ., . .. University of Ghana http://ugspace.ug.edu.gh - 93 - Table 16 continued Effect of various Relative Humidities on the rate of growth of' germ tubes)f P. corylea at 25°C. (Relative Humidity maintained with saturated aqueous salt solutions). - % E:xperi- Germ tube length in )l at Relative cent indicated hours Humidity Number 6 12 18 24- ----- - -j 61.9 62.5 61.2 64.1 I 2 72.0 75.9 67.9 69.0 33,2 3 60.6 68.5 67.2 68.6 ----... Mean 64.8 69.0 68.8 • 67.2 - -- : ,I ! , 1 I I I 50.8 i 55.6 I I 4-8.0 47.1 ~ 2 54.3 ! 59.9 64.0 58.1 12.0 3 50.5 I 53.8 63.4 57.4 Mean 51.9 56.4 58.5 54..2 ._- . I - 1---- -1 51.9 I 53.3 49.0 37.1 2 48.3 52.6 I 54..9 55.7 0.0 3 ! 52.1 44.8 50.0 51.4 i Mean J_50 •. ~ _ - 50.2 -~ 51.3 48.1 University of Ghana http://ugspace.ug.edu.gh •• 94- - 200 180 160 140 ~ ~ 120 :I: ..... I.!> Z w 100 -I W III X 75·8 % R.H. :;) ..... 80 -053·8 % R.H. ~ ~ w ----B 33·2 % R.H . I.!) z 60 c( .----.,2.0% RH. w ----()---- () 0.0 % R.H. ~ 40 20 6 12 18 24 TIME IN HOURS FIG.II. EFFECT OF VARIOUS RELATIVE HUMIDIES ON THE RATE OF GROWTH OF GERM TUBE OF P. CORYLEA AT 250C. University of Ghana http://ugspace.ug.edu.gh 95 - H. GrnUfiNATION OF CONIDIA OF P. CORYIEt>. STILL ATTACHED TO CONIDIOPHORE. (1"7) BrodilJ anCl. Neufeld (1942) ani Clerk and Ayesu-Offei/aftfJ1' exhaushve examination f'ailed to find any spores of the respective powdery miJ.del':';; }J:. polygoni and L. taurica germinating while still attac~:ed to the conidiophores. Cherewick (1944-) on the other hani found that Gonidia e- f £:. graminis freely germinated i!!!.i:E!. During the present studies it was observed that conidia populatiO:1J obta ined on slides occasionally included germinated ones. The proportion of' these, however, is extremely low (about one in a thousand). Such c~nidia might have two possible origins; either they were conidia l7hich :night h a.vt; germinat '3d E ~ or were detached conidia which had @9r~~ted on the host epidermis. The form~ is more probable since the diseased leSions occur only on the abaxial surfaces with the conidiophores hanging down. Detached conidia would therefore more naturally drop downward into the air. Several diseased leaves were therefore exam~ed under . the microscope for @arminating conidia. Indeed, some conidia, but ra~her scantily were observed to have germinated whilst still attached to the conidiophore. (Plates 14-, 1.5 and 16). It is highly probable that these conidia might have been stimulated under unkn?wn peouliar conditions, which University of Ghana http://ugspace.ug.edu.gh PLATE 14.: Photomicrograph of conidiull o~ P. cor.ylea at early stages of germination whilst st:i11 attached to the conidiophore. x 190 University of Ghana http://ugspace.ug.edu.gh - 97 - PLATE 15: Photomicrograph of ccnidium of P. corvlea at advanced stage of gerlllination whilst stUI attached to the conidiophore. I 190 University of Ghana http://ugspace.ug.edu.gh 98 - PLA1'! 16: Photomicrograph of conidiUIIl of P. cm-ylea germinating whilst stUI attached to the cGllidiophore. Note the absen::e 01' constriotion between conidium and conidiophore tip. X 190 University of Ghana http://ugspace.ug.edu.gh 99 - occurred even be~ore th~ spore was fully prepared for detachment (see pages 159 - 162). On the other hand, some of the detached conidia lying either directly on the epidermis or trapped in the ectotrophic ~celium were found to have germinated. In this case, again, the number of conidia doing so was low, involving about 1 per cent of the total number of detached conidia. University of Ghana http://ugspace.ug.edu.gh ":' 100 - I. SURVIVAL OF THE GERMINATED CONlDIA OF P. CORYLFA. It has been reported that germinated spores of some fUngi survive drying for oonsiderable periods (Calpouzos, 1955; Grindle and Good, 1961; Goos and Tschirch, 1962). This does not appear to be so in other species (Doran, 1962; Yarwood, 1936). It seems that the role of the spore dried after it has germinated, but before it has penetrated the host tissue is important from the standpoint ~f infection. If only a. small proportion of such spore survives a dry period, this m~ have considerable effect on the epidemiology of the disease. With P. corylea a large percentage of conidia (more than 5~~) germinate at even of, R.H. The germ tubes produced at the lower humidities were observed in the pr"ceding experiments to dry up quite readily. Spores under such conditions which do not permit long survival of the emerged germ tubes, may therefore have very limited value as infection units. This experiment therefore attempts to establish the longevity of' the germ tt bes umer the various humidities. Conidia on glass plates (42 x 42 mm) inverted over Van Tieghem cells (see , general method, page 16 ) holding saturated aqueous salt lJolutions to provide various relative humidities (see Tables 17 to 19) University of Ghana http://ugspace.ug.edu.gh 101 were incubated at laboratory temperature (27 ~ o1 C.) ~or a total periud of 48 hours. At 6 hours when all the germinable conidia had germinated, the respective percentage of germ tubss which have shri- ve1led at the varicus humidity levels were observed. These observa- tions were continued ~t the indicated intervals in Tables 17 to 19. In ~ach case percentage of gorminated conidia with shrivelled and twisted ~rm tubes were assessed. From this value was also calculated the percentage of 1::1e germinat",d conidia with unshrivelled germ tubes, a datt,; mused ' ,for 'thtl graph in Fig. 12. No further reading ~ar a particular s8.lLple was taken after -~l-te percentage of germinated conidia with Shrivelled twisted germ tubes showed 100% shrinkage for three consecutive observations. The results are presented in Tables 17, 18, 19 and 20 am in Fig. 12. Camera lucida drawings ' of representative germinated conidia at intervals of 3, 6 and 12 hours held at some of the relative humidities (0.0, 33.2~ 53.8, 75.8, 96.9,% R.H.) were made and illustrated in Figs 13 to 17. The results showed that th~ germ tubes shrh'elled quickest at 0% R.H., where all were dry after 12 hours. As the humidity increased., however, the rate of shrivelling of the germ tubes decreased. The germ tubes therefore remained turgid IOilgas'c at 100% R.H. At this humid ity about 80 per cent of the germ iubes were still fully turgid after 48 hcurs. University of Ghana http://ugspace.ug.edu.gh - 102 - TABLE 17: The rate o~ shrivelling ~ germ tubes o~ conidia of P.corylea incubated at 0.0, 12.0 and 33.2%R.H. and at 25°C. Time of Percontage of germinated conidia Incubation with shrivelled germ tubes at in indicated relative humidities. Hours o.~ 12.0% 33.2% 6 15.5 10.9 1.5 7 20.5 14.0 2.5 8 39.2 26.6 5.9 9 48.4 52.5 6.8 10 68.8 62.5 23.5 11 100 80.7 34.9 12 100 87.4 40.3 13 100 94.4 46.5 14 -* 100 55.2 15 100 69.6 16 100 83.2 17 93.2 18 100 19 100 20 100 21 .L - • No fUrther reading taken. University of Ghana http://ugspace.ug.edu.gh - 103 - TABLE 18: The rate of shrivulling of g(;rm tubes of conidia of P. corllea incubated at 53.8, 75.8 and 80.3% R.H. and at 25°C. Time of Perc~ntage of germinated conidia Incubation with shrivelled germ tubes at in ind icated relative humidities. Hours 53.8% 75.8% 80.3% _ . ___ .. _ww 6 1.0 0.0 1.0 7 4.4 0.0 2.5 8 6.0 3.2 8.3 9 18.0 8.9 15.7 10 27.8 11.0 14.8 11 31.0 12.6 15.0 12 33.4 17.7 19.3 13 47.2 32.8 26.3 14 49.2 3~.6 29.7 15 52.7 47.1 29.2 16 70.0 53.0 31.8 17 86.4 62.0 39.4 18 91.6 65.7 40.2 19 94.1 71.3 56.0 University of Ghana http://ugspace.ug.edu.gh 104- - Table 18 continued The rate of shrivelling of germ tubes of conidia o~ P. corylea incubatod at 53.8,75.8 and 80.3~R.H and at 25°C. Perc~n::;age o~ germinated conidia Time of with shrivelled germ tubes at Incubation indicated relative humidities. in Hours 53.8% 75.8% 80 • .3r~ 20 100 72.3 70.3 21 100 81.2 79.1 22 100 83.2 80.9 23 _i/) 85.0 81.1 24 95.0 85.6 25 98.0 90.0 26 100 92.5 27 100 97.7 28 100 98.1 29 I 99.0 30 100 100 31 l _32_ 100 • No further re-ad-:-:-i-ng-t-ak~e-n-.---...L------ University of Ghana http://ugspace.ug.edu.gh - 10S - '.rA.BLE 19: The rate of shrivel~ing of germ tubes of conidia of P. corllea incubated at 92.0, 9~.9 and 10q% R.H. and at 2SoC. Time of Percentage of germinated conidia Incubation with shrivelled germ tubes at in indicated relative h. .l lnidities. Hours 92.~ 96.910 10Q'fo 6 0.0 0.0 0.0 7 3.0 0.0 0.0 8 4.9 0.0 0.0 9 9.1 1.5 0.0 10 9.S 2.5 0.0 11 10.0 3.9 1.0 12 12.0 5.0 2.S 13 20.0 5.5 2.5 14 23.2 5.7 2.7 15 25.5 6.0 4.0 16 27.9 7.5 4.0 17 37.4 10.0 4.9 18 42.7 14.0 5.0 il20 ~·2 16.0 5.0 50.2 16.9 7.5 21 61.8 17.0 --_ 9.5 ........... University of Ghana http://ugspace.ug.edu.gh - 106 - Table 19 continued The rate of shrivelling of germ tubes of conidia of P. corylea incubated ~t 92.0, 96.9 ani 10Q% R.H. and at 2SoC • . -- - TimE:! of Perc~ntage of germinated conidia Incubation with shrivelled germ tubes at in indicated relative humidities. Hours 92.0% 96.970 1 OOJ~ 22 66.1 . 17.1 9.5 23 67.6 I 19.0 10.0 24 68.7 20.0 11.0 25 70.2 21.0 11.0 26 72.8 21.0 14.0 27 79.2 22.5 16.0 28 80.0 22.5 18.0 29 81.7 22.9 18.0 30 82.5 23.0 18.5 31 90.5 23.5 18.5 32 91.6 23.5 19.0 33 93.8 24.2 20.0 34- 93.8 24.2 20.0 35 93.9 24.6 20.0 36 94-.0 24.9 20.2 ~_. ..l 94-.5 25.0 20.2 University of Ghana http://ugspace.ug.edu.gh - 107 - TABLE 20: Percentage o~ germinated conidia with unshrivelled germ tubes at various relative humidities ani a.t 25°C. (Relative Humidities maintained with saturatud aqueous salt solutions ). ---_.--- --- -- % Time of incubation in Hours Relative - Humidity 6 12 18 24 30 36 - 100 100 97.5 95.0 89.0 81.5 79.8 96.9 100 95.0 86.0 80.0 77.0 75.0 92.0 100 88.0 57.3 31.3 17.5 6.0 80.3 99.0 80.7 59.8 14-.4- 0.0 0.0 75.8 100 82.3 34-.3 5.0 0.0 0.0 53.8 99.0 66.6 8.4- 0.0 0.0 0.0 33.2 98.5 59.7 0.0 0.0 0.0 0.0 12.0 89.1 12.6 0.0 0.0 0.0 0.0 0.0 84-.5 0.0 0.0 0.0 0.0 0.0 ----- . University of Ghana http://ugspace.ug.edu.gh .>..' .J ~... • ;"" ~ 0 'f.0 0 b .,o~~/O ,-a: "'>$ ~,O~· ~O • G'tlf6'" ~ I~C' ,. • ~O ot'''' (t~. .1 ': 1-" \~\ 10~ I~ ~() ~$ ~~,O'~ "'0ll. "$. ~,-It. fIo.t."'" FIG.12. PERCENTAGE OF GERM TUBES OF P. CORYLEA CONIDIA VIABLE AFTER STOR'·ACG,E AT DIFFERENT RELATIVE HUMIDITIES FOR VARYING PERIODS AT 27± University of Ghana http://ugspace.ug.edu.gh 109 _ J HOUAS AFTEA INCUBATION. F/G . /3 . CAMERA LUCIDA DRAWINGS OF GERMINATED CONIDIA OF ....e,CORYL~ GINECRUMB ATTUEBDE AST. 0 '0-'0 R.H. AND AT 2S-C . SHOWING RATE OF DRYING OF University of Ghana http://ugspace.ug.edu.gh FIG.I •. CAMERA LUC IDA DRAWINGS OF GERMINATED CONIDIA OFU OeYL€A. INCUBATED AT 33 ·2 °'0 R.H. AND AT :lS·C. SHOWING RATE OF DRYING OF THE GERM TUBES . University of Ghana http://ugspace.ug.edu.gh - 111 - 12 HOURS AFTER INCUBATION . FCIAGM.IESR. A LU C I OA DRAWRINGS OF GER"'I~ATE Ho OC H AND AT 25 C.$ WOINNGID IRAA TOEF OPF C DORRYYLINCG;A OF INCUBATED AT 53" "/0 .. THE GERM TUBE!>. University of Ghana http://ugspace.ug.edu.gh ,' ~..,1<0~M .~ER1'&o. ''"; :\!.U ~c.~~';" :O."R ... WI~GS Of GERMI~"'Tf.O C.O~IOI'" Of P. c.ORY\.E'" IO ,. ,.," We. ,.0"·' .,,' " •• ".&01 University of Ghana http://ugspace.ug.edu.gh fIG .17. CAMERA LUCIDA DRAWINGS OF GERMINATED CONIDIA OF P.CORYLEA INCUBATED AT 06·0·/0 R.H. AND AT 25°C . SHOWING RATE OF DRYII"G OF THE GERM TUBES . University of Ghana http://ugspace.ug.edu.gh 114 - It was als~ observed that slight wrinkles appeared on some of the germ tubes and the conidia but these did not show clear visible shrinkage and these were therefore excluded from those judged shri- velled. If those in reality were showing a stage or another form of shrivelling, then the values of p~rcentage of shrivelled germ tubes might be slightly higher by about 4 - 6% Of the ,"xpressed figures in oach ca se. The results also showod that shrivelled twisted germ tub es were observed even ~t 10Q% R.H. as early as 11 hours after incubation. This shrinka~ of g~rminated conidia at 10qrb R.H. probably shovied death of the germinated conidia through another cause ani not by actual loss of water. On the whole it was noticdd that the longer germ tubes collapsed er~rl:isr than the shorter germ tubes, perhaps duo to exposure of greater surface area to the influe- nce of the humidi~. Throughout this discourse it has been implied that Shrinkage is synonymous to death. Since certain conidia are known to r~cover when r~ turned to favourable conditions after they had boon desicoated for some time (Grinile and Good, 1961; Goos and Tsohiroh, 1963) the shrivelled conidia of P. corylea could only be described as dead if they failed to l'egain their turgidity if placed at higher humidities. In eaoh caso, thert:fore, where 100 per cent shrinkag€: was achieved during these studies, m., conidia at 0.0, 12.0,33.2,53.8,75.8 University of Ghana http://ugspace.ug.edu.gh 115 - and 80.3% R.H., th0 conidia were transferred to humidity chambers of 100% R.H. , and obsorved after 12 and 24 hours. In none could any r e.::overed germ tube bt:; found. University of Ghana http://ugspace.ug.edu.gh 116 - J. PATTERN OF G-ERMINATIOrJ OF CONIDIA OF P. CORYLFA The pathrn of germination of the conidia of some powdery mildews has been studied by 1IlIlny' workers and cbservations are <:pite varied. Brodie am Neufeld (1942) showed that in germinating conidia of E. polvgoni the germ tubes emerged excentrically from one em of the conidium and never directly from the em or from the sides. ~imilar behaviour was noted in germinating conidia of LeTeillula ~ by Nour (1958) and Clerk and Ayesu-Offei· (1967). Yarwood (1957) stated that this mode of germination was common to the Erysiphaceae in general. The latent period of germination .:1180 seems to be quite uniform among the powdery mildews. Brodie ani Neufeld (1942) indicated that germination starts within 2 hours. 57) respecti valy found similar behaviour in uredospores of crown University of Ghana http://ugspace.ug.edu.gh rust of oats am conidia of Monilinia fructicola. Nagvi and Good (1957) reported that th" conidia of Monilinia !r.£,.cticola. survived longest at 75~ R.H. Other investigators for example, Merrick and Fergus (1954) found thr.t at 12 - 24°C., the endoconidia of Endoconi- diophora fagacoarum remained viable longer at 95% than at 75% R.H., am Goos and Tschirsch (1962) r<3portoo. that spores of Gloeosporium ~ survived longest at higher humidities (60 - 80% R.H.) tha:n at l")wer humidities (0 - 2~ R.R.). A fourth type of relationship was reported by Teitell (1958) who found that within the range of atmospheric humidities that Was too dry to permit of germination, survival of Aspergillus ~ conidia was longest at low (~R.H.) and high (85% R.H.) humidities and briefest at a narrow intermediate range clcse to 75% R.R. Rtl found a similar rela tionsh ip also for an isolate of A. terreus. A possible third example of this sert of relationship is provided by the ascospores of Endooonidio- phora fagacearum. Although Marek and Fergus (1954) interpreted their results on longevity of ascospores of this species as indicating longest rurvival at the lowest humidity and least at high, they did in fact show that between 12 and 24°C. all the University of Ghana http://ugspace.ug.edu.gh 137 - ascosporos died in l~ss time at 75% R.H. than as 50, 25 and 10Jb R.H. Conidia of Metarrhizium anisopliae, appr:C'.Xs to be what is so far only the fourth known example of a fungus with spores surviving for the least time at a m~ian humidi~ (c. 45% R.H.), and longest above and below this 1 vel (Clerk ani Madelin, 1965). The effects of light and temperature on spore lcngevity shows less variation. Almost witmut exception, survival of fungus spores is gr~a.test at lew temperatures (Cochrane, 1958) ani in spores sensi ti ve to light, their viability tends to be shortened by light; this effect being more mar1o:;,d in hyaline than in coloured spores, '/l ith P. corylea. conidia which readily germinate at an,y humidity level at an,y favourable: tempera .ture, longevity studies could only be carried out within a limited r::mge of temperature, that is, between freezing and the minillUm temperat·u.re permitting germination. This was th ,) condition, ther0fore, urrler which the longevity of th" conidia was examined. Conidi~ were stored on glass slides over respective saturated aqueous salt solutions (which me.intained ccnstant relative humidities) in humidi~ ohambers soaled with cello tape , in darkness in a refrigerator set a.t SoC. l\nhydrous calcium University of Ghana http://ugspace.ug.edu.gh 138 chloride 3.nd water were used for constant humidities of O]b and 10q% R.H. raspectively. Viabili~ was determinud after desired intervals of storage by transferring ani germinating the conidia. at nOc. and at 100% R.H. for 24 hours. Perctmtage germination was then determined and the 10ngth of germ tubes measured. /hen in the longevity test there was no germination in a :Jample, :urther sample s were taken fi'om the same vessel at th 0 next ~o sampling dates and tested for g~rmination. If still negative, the pcpulation was prusumed wholly to have lost thewility to germinate and no further samples were taken. Tne data of the results (Table 23 and Fie. 24) show clearly that at even such a low tempera.ture of SoC. , the conidia of P. corylea were ' poorly pres€rved. In no sample did longe- vity exceed 20 days" Conidia of P. corylea were ve~ short- lived at low humidities and loss of ability to germinate was extremely rapid in conidia stored at 016 R.H. J so that by the fifth day there were no viable spores. Rising humidity increased longevity and the longest survival lias at 82.6 to 10Cf~ R.n.; bet-ween 0.2 and 0.4% conidia were still able to germinato after 1S days. University of Ghana http://ugspace.ug.edu.gh - 139 - TABLE 23: Percentage of conidia of E:.. corylea able to germinate after storage in darkne s s in atmospheres of various humidities at 5°C. (Percentage germination at 1 OCl}'~ R.B. aft~r 24 hours at 27°C.) % Period of Storage in Days Relative 1 2 3 Humidity Mean Mean %G er~- ~atbe %G e~i':' ~ tul:s ~'fube nation ~ength in nation length " inp na Gtieornm i- length )l in)! 0.0 13.6 114.1 4.1 72.5 0.9 45.7 14.0 14.8 140.0 5.6 37.7 1.4 47.6 34.6 10.9 109.0 9.6 98.8 1.8 21.5 59.2 11.8 92.1 11.2 103.0 3.5 70.9 75.1 12.4 117.3 9.2 87.7 3.3 56.3 82.6 15.1 119.8 5.3 90.0 4.0 90.4 96.6 11.5 I 121.2 4.8 105.5 6.0 89.8 100.0 9.6 115.9 4.9 94.7 1.6 86.4 University of Ghana http://ugspace.ug.edu.gh - 14-0 - Table 23 co ntinued percenta.ge of conidia of P. corylea able to germi.nD.te after storage in darkness in atmc.lSpheres of various relative humidities at 5°C. (Perccnt~ge germination at 100% R.H. aftdr 24 hours ~t 27°C.) Period of Storage in D:>.ys Relative 5 6 Hu mi d't l. y % Mean Mean Garmi- r Mfge rm t euabne %G :ermi- germ tubo %Germi- germ 'b.ile 10ngth length "'. hon length In ation nation : ---- in)l , i-np- in? ,, 1--- 0.0 0.6 19.6 I 0.0 I 0.0 14.0 0.3 98.0 I 0.4- 24-.0 0.5 24.5 34.6 0.7 56.0 I 0.5 18.2 0.5 28.0 59.2 4.2 106.8 0.1 24.5 0.1 20.2 75.1 5.9 88.7 2.4- 82.6 0.9 49.0 82.6 4.0 90.4- 4-.5 74-.1 1.0 35.0 96.6 6.6 110.6 2.6 61.8 1.2 35.0 :00.0 4-.5 100.9 3.5 68.2 1.0 38.5 University of Ghana http://ugspace.ug.edu.gh - 141 Table 23 contirued Percentage of conidia of?-:- corylea able to germin~te after storage in d~kness in atmospheres of various relative humidities at 50 C. (Percentage germination at 100% R.H. afte~ 24- hours at 27°C.) Period of Storage in Days % Relative 7 8 9 fumidity Mean Mean Mean % GermS· ge:"m tube %Germi- germ tube %G erm i- germ "tube nation length nation length nation length in? in)l in)l I 0.0 I 0.0 - .- I I - - - I I 14.0 0.4 I 2+. 0 : 0.2 ! 17.5 0.0 i ! i - 34.6 0,6 I 20.0 0.4 11.6 0.2 11.8 I 59.2 0.1 18.0 0.0 - 0.0 - 75.1 0.4 I 41.9 0.6 12.3 0.4- 50.4- 82.G I 1.2 36.1 1.4- 32.6 0.4- 63.') 96.6 1.3 ! 36.1 1.4 35.0 1.2 54.0 t 100.0 2.0 I 60.0 I 3.7 50.1 1.5 58.8 I I University of Ghana http://ugspace.ug.edu.gh - 142 - Table 23 continued Percentage of conidia of P. corylea able to germinate after storage in darkness in ~tmosphere8 of various relative humidities at 5°C. (Per~entage germination ~t 100% R.H. after 24 hours at 27°C.) Period of Storage in Days %0 10 15 20 Relative ---- Humidity Mor.n Mean Mean ~~ Germi- germ 'blbe %G ermi- germ tube %G ermi- germ tube n~tion length nation length nation length , in)l in )l- in? 0.0 - - - - - - 14.0 I 0.0 - I 0.0 - - - 34.6 0.0 - 0.0 - 0.0 - 59.2 0.0 - - - I - - 75.1 00 4 ( I 14.0 0.0 - 0.0 I - ! i I 82.6 ,1 0.4 54.5 0.4 17.5 0.0 I ! - 96.6 1.2 68.7 0.4 13.0 0.0 - 100.0 0.4 67.7 0.2 17.0 0.0 - University of Ghana http://ugspace.ug.edu.gh 1&.1 C) ~ Z 1&.1 ..... U cr -$. A'". ,o~-9,O ~ >$ c;E. . Ofl-~ °0~ ~1". " ,0 ~O Of ~"<. O~ \0\0' '4c:~ , ~ • "<.0( 'IV () '\ ~S ..., 0' '4"~. +\l .... ~,-aE. ",O~O fi.E.",t- FIG.2". EFFECT OF RELATIVE HUMIDITY ON LONGEVITY OF CONIDIAOF..J!£QE..YL~ INCUBATED AT S-C. University of Ghana http://ugspace.ug.edu.gh 144 - M. DEVELOPMENT AND RELEA...'m OF CONIDIA OF P. CORXLEA. The follo)/ing expericent s were conducted to examine two aspects of the biology of P. co~lea; these are, the morphology of the conidiophore ani formation of the conidium and the malL'1er of rcleltse of' the mature conidium. The stntct-ure of' powdery mildew conidiophores is sometimes confused by the ~~~zi~ vRriation in the same species on the same host as observed by different investigators. For example, the illustr:l.tion~ of conidiophores cf !lP~ ~ from grape by Vip..!_a (1893) and Cooke (1905) in }~urope ani by Galloway (1895), Longy-ear (190l~) and Duggar (1909) in the United States I'l.re t'J. ccc:'ly dlf'ferent from each other y0t all are generally regarded as of the saiJe species. Whether these differences rGP1~esent truly different fungi or ,;h.::ther they are differences due to cnvironment, host variety or differences in the illustrating ability Cit the observers cannot be deter- mined at this time. Further examples with the sa~ sp<;cLs, as well as with others, could be given. On th& other hand, uniformi~ in reported structure is ==== C>= IIAM .~ ~ == ~ 12 PM . ~ ~ = I PM. ~ ~ '-----' 100)J, A B FIG.33. CAMERA LUCIDA DRAWING$ 0' DEVELOPING CONIDIUM OF e.CORYLEA AND ASSOCIATED BENDING 0,. THE GENE- TIVE CELL AT 20· C . University of Ghana- http:/-/ugspace.ug.edu.gh185 J' DATE TIME < 29:3:688AM. e 30:3:667AM . ~ 8AM. c:::=r C::::' .. 9AM . C:::' C::' lOAM. C:! C> == IIAM. C":> 0: --==- " 12 PM L r " I PM . ~, 0:. A B FI(, . 34 . CAMERA LUCIDA DRAWINGS OF DEVELOPING CONIDIUM OF P.CORYLEA AND ASSOCIATED BENDING OF THE GENERATIVE CELL AT 28°C. University of Ghana http://ugspace.ug.edu.gh 186 - b. EFFECT OF MOISTURE ON rooolING OF 'l.'HE GENERATIVE CELL If moisture plays a role in the bending mechanism, that is, too generati va cell either loses or takes in water dur in8 berding this MY be accordingly Ilffected by different moisture levels. Conidiophores on diseased leaves were again kept urder investi- o gation in the usual manner in the laboratory (tempernture 28 e.). Two experiments were set up. One experiment was under a plastic hood which had previously been sprinkled with water to provide an atmosphere of 10010 R.H. 'l.'he other remained uncovered ani was placed near a table fan to keep the: humidity slightly lower than atmospheric humidity (atmospheric humidiw at time of experimenta- tion 76% R.H.). The conidiophores were again observed ~t hourlY intervals aft .~r th e conidia h:>.d d eveloped and tre berding waS recorded by menns of camera lucida drawings. The hood oVlolr tho microscope was lifted for drl'.w:ings to be made a.ni quickly put back. Generative cells uro conidia of P. corylea, the major infection units, have been studi"d in addition to some aspects of the biology of this fungls. The climatio factors aff&cting the germinr1.tion of the oonidia studied here e.rG tel!!perature, humidity and light. Temperature relation of the powdery mildews has been rf..'Viawed by Yarwood, Sidq and Cohen (1954). In their revhm, it was reported that the optillUm temperature for powdery mild <:ws ranged from 11 to 28°C. depending on the species and th R.H. of 79.4 per cent observed in one of the exp~riments (see Table 11) seems to be thlOl highest germination at this humidity racorded among the Erysiphaceae. No particular humidity level could therefore be considered as cptimum with regards to conidial germination in P. corylea since germination was so uniform and even the rates of germination (see Table 15) were similarly identical. It was however possible to establish this by the amount of germ tube growth supported. Germ tubt;s weN always longest at 10q1;R.H. (99.1 - 138.4,u) and shortest at o,;.R.H. (17.3 - 27.2)1) (see Tables 8, 10 and 11). University of Ghana http://ugspace.ug.edu.gh - 2Q4. - 'Nith species such as P. corylea in which lom,r l'umidities permit sufficiently high conidial germination, cons:idera.ble infection units could be mltbilised umer a wide range of atmos- pheric Immidity corilitions. Many Y,orkers claim that in general the powdery mildews sp~ad rapidly unier dry climatio conditions (Volk, 1931; Honecker, 1936; Cherewick, 1~; Last, 1955). This is however not always so, for the powdery mildew, L. taurica has been observed to occur abumantly in the wet season in Ghana and very sparsely in the dry ,eason (Ayesu-Offei, 1966). The ability of the conidia of P. corylea to germinate very well at extremely low humidities probably explains the persistence of the disease on pawpaw plants throughout the year, ani flourishes in the rainy as well as in the dry (h",rmattan) seasons in this country. :furtherlllOre, the conidia of p. corylea could germinate very well under fluctuating atmospheric Immidities, thus sdemingly well suited for its rol~ as infectum units. The virtually unique property of germination at zero relative Immidity confers on the powdery mild ew fUngi considera- ble theoretical importance. This ab ility to ::;erminate at very low relative humidities has received two interpretations. Comparative studies of the water content of' most fungal spores i8 low. For exampl&, Yarwood (1950) found that the water content University of Ghana http://ugspace.ug.edu.gh 205 - of spores of Penicillium digitatum and Aspard.llu~ niger were only 6.0 and 13.0 per c,mt respectively. Yarwood, 011 the other hand, found that the ;/ater content of conidia of th,· ~)o.;'tiery mildews E. polygoni, E. gaminis and E. cichoracearum raIl6 ... d from 52 to 75'J: of th eir £'rush weibht. Apparently, the low we.t.r content of most fungal S~l()res demarrls prusence of tJxt"rnal water for germina- tiona Yarwood (1950) suggested that the high water content of powdery mildew conidia, on the other ham, may "xplain their ability to ~rminatG at low relati~e humidities. He argued that there is enough water in the conidium to initiate g,.rmination. His observa- tions of shrinking conidia of the three sp~ias during germination at low humidities \ 13.5 put forward to support his claim. The shrink- age of tht:; thin delicate walls of the conidia ·.vas thought to be due to fall in ,;ater l evel :in the conidium. Yaruood (1952) working again on E. grminis substantiated his earlir;r propositiGlns. He observt;d that ",fJrminating conidia lost mort:! w·\ter than non- germinating conidia and that while both germinatin.::, and non- germinating conidia shrank in 9. dry atmosphere the ,germinating spore shrank at a fast 'cr rate. Brodie and l'reufeld (194.2) had earliar presented an alternative int·,rpretation. " 'h"y oontended that the /Jl£.ture conidia of !a....J2gJ.ygoni and E. graminis contain ve~ little water bofore commencement of University of Ghana http://ugspace.ug.edu.gh 206 gerr..ination but water is released through changes in colloidal materials oontaining bound water during respiration. They claimed that no shrinkage was observable in conidia germinating at the l~wer humidities. Brodie (1~5) fUrther suggested that the high osmotic pressure of the conidia of E. polygoni ani !j. sraminis could enable them to absorb water from relatively dry atmosphere, hence their ability to germinate at these humi- di ties. It is however highly improbable that conidia will tim sufficient moisture to absorb at extremely low humidities and this hypothesis fails completely to account for germination over desiccants (zero per cent relative humidity) where the atmosphere is completely devoid of moisture. Observations made in the present work favour Yarwood's (1950) h;ypothesis that the source of water for @rmination of the conidium of the powdery mildew at low humidity is stor ...'.< l water in the conidium. F. carylea conidia at any humidity germinated to the same d&gree, probably dep.::nding entirely on emogenous water supply. On the other ham, those at the h~her humidities showed longer germ tubes, may be sustained by external moisture whilst those produced at the lower humidities remained srort because of lack of additional water supply from the atmosphere. University of Ghana http://ugspace.ug.edu.gh 207 - Besides, germinating and ron-germinating conidia of p. corylea did not sbril''\( when incubated at 1 00% R.n., whilst varying percentages of the con:idia. shrank at relativtl humidi- ties below 10010 R.H. The percentage of conidia shrinking at the higher humidities was small and increased at th0 lower humidities (see Table 8). This shrinkage has been accepted as evidence of loss of water, a hypothesis consistent with observa- tions here. Yarwood (1952) has ruled out the possibility that a volatile substance escaVing from the spore might b& the causa- tive f'actor of this collapse. He argued that this then could have taken place at lower humidities as well as 10~; R.H. Most probably the germinating conidia obtained abundant external supply of wat&r at the higher humidities, and need not be sus- tained solely by the internal water. It can be concluded from results of this work that the shrinkage may bci due botbto loss of watet' by the spore to the air, especially in non-germinated conidia, and to use by the con:idium for germination activities. Yarwood (1952) did indeed observe that germinating con:idia of E. polygoni showed a greater decrease in volume than shrunken non-germinated conidia. The physiology of germination of the spores has rot b&en studied, and until this is done, knowledge of m~tabolio activities involved in germination of powdery mildew University of Ghana http://ugspace.ug.edu.gh - 208 - conidia will be a matter for conjecture. Many workers (e.g. Grat-Marin, 1934; Yarwood, 1936; Clayton, 1942; Grainger, 1947) have stated that ~rmination of conidia of the Erysiphaceae is poor at 10~ R.H. and that the optillllm relative humidity lies at a level slightly below satura- tion. The pres snt ob 3ervations (s~e Tabl€s 10 and 15) and the r esults of Nrur (1958) on L. taurica ani those of Mamers am Hossain (1963) working on E. praminis am of Ayesu-Offei (1966) also working on L. tauricp. ind:icate that 100% R.H. is th.:; most favourable !bvel for germination of thase conidia if' car" is taken to exclude liquid water. It is only when condonsation occurs copiously that poor germination obtains. A supporting evideme is providrus arcularis and Chaetomium ,ionesii germinated norma.lJ.y over the parent colonied. The behaviour of conidia cf P. c .. rylea ma;y be due to one of two causes or both. The conidiophores either produced diffe- rent levels of an inhibitory factor with age; the factor being in low concentratio~s at tne early stages of spore development per- mitting some germination and then increasing to inhibitory levels at last stages of spere development qr produced toth inhibitory and stimulatory factors together in different proportions at different stages of spore d~velopment. The spore ma;y then germi- nate er remain quiesoent depeming on which factor is predominant. University of Ghana http://ugspace.ug.edu.gh - 222 - VI. SUMMARY 1. Conidia of P, corvlea geminated at all the five tempera- tures viz., 15, 20, 25, 30 ani 35°0., used. 2. The respective mean percentage germination at 15, 20, 25, 30 and 35°0. nas 10,8, 22.0, 60.6, 47.7 ani 4.9 per cent. in 24 hours. 3. Althvugh th" conidia germinated best at 25°C. tha germ tubes at 30°C. were slightly longer than those at 25°C. 4. The optimum temperature apparently 183" between 25 ani 30°C. 5. Only 10,0 per cent of P, corylea conidia germinated in liquid water at 25°0, 6. Conidia imubated at 25°0, showed thll respective mean percentage germination of 41 .0, 44.1 , 46.1 , 45.7, 46.0, 49.5, 50.6, 50.4, 48.2, 44.5 and 52.3 at humidities of 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 and 0% R,H. 7. At 25°C, and at respecti~e humidities of 100, 96.9, 92.0, 80.3, 75.8, 53,8, 33.2, 12.0 end ~ R.H., 56.9, 54.2, 53.4, 48.5,45.8, 52.6, 54.7, 51.5 ani 50.5 per cent of the conidia germinated. University of Ghana http://ugspace.ug.edu.gh - 223 - 8. Conidia incubated at 27 .:!; 1 :>C. attained the r ospective percentage germination of 81.1, 92.9,82.5, 71.2, 76.1, 71.0, 79.4, 68.2, 70.4, 75.0 and 72.3 at humidities of 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 and or~ R.H. 9. Conidia exposed to £'1uc'lllating atmospheric relative humi- dities of 64.0 to 89.a;b R.H. durin[; initial perWl of germination showed 63.9 to 80.4 per cent germination. 10. The rate of germination was uniform over rumidity range of o to 100}6 R.H. and the latent period of germination was 2 - 3 hours for each of these humidities. 11. The germ tubes grew more rapidly and longer at 100% R.H. than at any other humidi 1;y. Lengths of germ tubes dimini- shed with decroase in r ela tiv e lumid ity • i2. Equal proportions of germinated and ungerminated conidia shrivelled at the various relative humidities. 13. Appressoria formation followed no definite pattern, iIrlica- ting that relative l'D.lmidi ty has no inf'luencl;) on apprl;)ssoria development. 14. The g~rm "lubes of P. corylea. emerged more frequ ently but raniomly from the club-shape end of the conidia. 15. Only a single germ "lube usually emerged from each donidium ani less frequently t ·,IO. University of Ghana http://ugspace.ug.edu.gh 224 - 16. Branching of germ tubes on glass surface was rare, and :if' present was not extensive. 17. The conidia germinated at both &1rfaces of the host (C. papaya) leaf and tho germ tubes grew randomly ove~ the host leaf surface. 18. Germ tubes shrivelled q).lickust at afo R.H. and the rate of shrivelling of germ tubes decreased as the humidity rose. 19. Conidia germinated bot"Wr and produced longer germ tubes though fewer appressoria in light than in dark. 20. Conidia of P. corylea lost viability most rapidly at the lower humidities and were preserved longest at the higher humidities. Conidium longevity was however very brief. The l~ngest surviving conidia stored at 82.6, 96.6 and 10~ R.H. lost viability in 15 - 20 days. 21. The conidiophoras ar0 averagely 3oo)l lvng, septate, 2 - 3 celled, unbran'}hed and arose from the ectophytic hyphae at right angle to tile leaf surface. 22. The first conid:illm of each conidiophore was cut off from a terminal erect generative cell. 23. Immediately after the conidium had fully matured th\;l gene- rative cell began to bem across the middle am which oontinued until the two arms ''If the generative oell stood apprOximately at right angles to each other. University of Ghana http://ugspace.ug.edu.gh 225 24. EUll maturity of the conidium was indica. ted by the s:iJlul- tanaous rounding up of the distal wall of the generative cell and the basal wall of thi;; conidium. 25. Duri..'1g bending of the generative cell, thRT, H. (1926). Factors affecting the development of flax rust Melamspora lini (Pers.) Lev. PHYTOP~TH. 16: ' 185-205. ' - University of Ghana http://ugspace.ug.edu.gh ... 233 - HAWKER, L.E. (1950). Ph;ysiology of' Fungi. 360 pp. UN!'Vr::RSITY OF LONDON PRlSS LTD. , WARWIC SQUARE, LONDON, E.C.4. HEBERT, T.T. AND A. KELMAN (1958). Factors u:nuencing the germination of' resting sporang~a of' Phys¢erma maya. is. PHYTOPATHOLOGY, 48: 101-110. HECTOR, G.P. (1927). 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Effect of' temperature and humidity on conidial germination in Erysiphe graminis. TRANS. BRIT. MYCOL. SOC., 46: 225-234. MARSH, P.B., E.D. T.t~:LOR .h.ND L.M. BASSLER (1959). A guide to the l~terature ")n certain effects of light on fungi..: reprodu ction J morphology pigmentation ani phototropic phenomena ' PL. DIS. REPTR. SUPPLe J 261: 321-351. University of Ghana http://ugspace.ug.edu.gh - 234 - McTEAGUE, D.M., S.A. HUTCHINSON MD R.T. REBD (:959). Spore germinati'm in Agaricus ca%iestrl.S L. ex. :me NcTURE, LOND , 183, 173 • McLAUGHLIN 'il D AND R.P. TRUE (1 952 ). The effec ts of temper a- , • ture and lnlmidity on the longevity of conidia of Chalara guercina. PHYTOPATH.,~: 470. MELHUS, I.E. (1915). Germination ani infection with the fungus of the late blight of potato. WIS. i~GR. 3XP. STA. RESEARCH BU:'L. , 21: 64 pp. MEREK, E.L. AND C.L. FERGUS (1951.. . ). The effect of temperature ani relative humidity on the longevity of spores of ihe oak wilt fungu s. PHYTOP1.TH.,!i!: 61-64. 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