A 57qu 0F LABORATORY METHODS '50 DETERMINE STRAWBERRY mam“. RESISTANCE TO GREY MOLD (30mm: CENEREA) 112 566 __THS Thesis for {‘[w Degree of M. 5. MECEIGAN STATE UNIVERSETY Thomas Bruce Ervine 1959 1n£§320 ll Ll! mu; mzuymwl Lu! (m 1111:5th mm m M II .FEB 0 6 7336 .f 5. ‘ .45. In. fl 1 4—9 cm""” if '11:” 13i-r- -~ 1r ‘ *1m r71 ‘\ ‘F'I‘lI ~‘1 hr "'l'_)! fij‘u‘fivir UL L11)... 03' IL.) L1-.. in; ¢-L+--ODS .LO LAAQ‘JL. .I-ab 3‘14““ 44.4-»:t— V51 T JTF RESISTAHCE TO G23 LOLD C OTfl"mle CILETLA) Thomas Bruce Irvine U) A TLESI Submitted to the College of Science and Arts of Lichigtn State University of.Agriculture and Apfilie( Science in partidl fulfillment of the rccuiremebts for the degree of LASTER OF SCIEKCE Depar ment of Botrny and Plant Pathology 1959 ETlUbCT 1 1 An investigation of technicues that coulc be em- ployed in a labor zto ory scree1in" test to evslurte rel€.- tive ver'etel fruit susceptibility to Urey hold (Eotrvtis eineree) wss undert: hen. Strawberry juice was extracted en: sterilized by Seitz filtration. These strawberry juices were incor- pore ted in both licuid en solid media and evelusted for growth of Jotrvtis cincree. Growth responses were consistent for certain “tr wberr 3r verieties throughout these methods. Comps ing pH an: percent soluble solids of the juice: with the growth responses obteized in the petri fl plete, growth tube and shake culture methods or evelue-- tion indicated with the exception of 3-2, which SUpport- ed Tetrvtis under the petri plate method only, all ver— (1) p-41 ieti 1 vin, a pH below 3.0 consistently foiled to 9) support Botrvtis in ell three swet: mols. Ho correlation was indicated between results obteined and percent sol— uble solids, or varieties hrving 3 pH above 3.0. Studies of spore ger1inet :Lon using hanging Cree preperations of juice di rectlv extracted from berries and inoculated with botrvtis spores was undertaken. An attempt wes nede to deter i11e tr e irtluesee 0:1 net- uritv or ste go or ripeness of the fruit ens variety upon the gimoz th of the germ tubes of ;. cineree. Tests of corre :tion between results obtained in uice Ol ripe 1ruit one (1—). the perm tube length in the the RVCC icl eenetrstion of whole ripe fruit gave a highly significrnt correlstion coefficient. It is suggested thst the spore germ inetion rethod be used as s pr li--n ry screening test in a breeding program fol- lowed by whole fruit penetration s udies lrter in the program. Add itionsl te sti11g in subseeum 1t see sons should disclose whether a definite pattern of TGS_DODS e cxis ts. TALLE OF COQTBgTS Introduction --------------------------------------- 1 Review of Literature ------------------------------- 2 Lhtoricls sud Kethofls A. Preprretion of Juices ------------------------- 6 b. Solid ledia a. Petri Plate Lethod ------------------------ 6 b. Growth Tube Lethod ------------------------ 10 C. Liquid Ledia a. Shske Culture hethod ---------------------- 10 b. Spore Germinction Lethod 1. Greenhouse Grown Berries -------------- 12 2. Field Grown Berries ------------------- 1% D. Whole Fruit Inoculation Lethod ---------------- 15 Results A. The pH and Percent Soluble Solids of the Strcwberry Juices ________________________________________ 17 n. Petri Plate, Growth Tube enu Sh"ke Culture ---- 19 C. Spore Germinction a. Greenhouse Grows berries __________________ 21 b. Field Grown Berries —————————————————— --_-_ 23 D. Growth 0? Isolates of Eotrytis cineree into tnole Berries --------------------------------------- 23 Discussion _________________________________________ 30 Summary ____________________________________________ 34 Bibliogreuhy' ------------------------------- 36 ILTLOLCCTIOH Grey Lold fruit rot, Ectrvtt; cincree, inflicts losses estinrtcd at over #130,000 a yecr to the Lich- igzn strevbcrry grocer (6). Control of this fruit rot bv funficidcl spreys hrs inyrovefi greatly within the nest ten yes 5, but spreyin" is costly Ind if improper- t ined is ineffective. The control of Grey Lold by varietal resistrnce is one of tle ultinrte goals of the strrwbcrry breeder. Evaluation of vrricties, selections and seedlings to Grey told resistance by field plot observations hes been inconsistent in the pest, prrtly due to the veriebility of the micro clinrte end the inoculum potentiel. Thus an investigation of techniques thet czulfl be employed [.4 J J C) eboratory screening test to evaluate reletive varietal fruit susceptibility to Grey Lold fruit rot was promoted. REVIEW OF LITERATURE Grey Lbld fruit rot, caused by Botrytis cineree, was first described on strawberry by F. L. Stevens in 191% (8). Kore than 100 different host plents have been reported for Botrytis cinerea (2). It may be found on all sorts of decaying vegetative matter and on the mulch and old lerves of the strawberry bed in an sea- son of the year (1). On strawberry, Grey hold attacks flower bud ped- icels, stems and celyces of small green fruit and leaf petioles (11,12) and it is not unusual to find an en- tire fruit cluster infected at once (1). Besides caus- ing heavy loss in yield, such infection builds up a high inoculum potential which under conditions of high humid- ity and temperatures around 600? (6) will invade the ripening crOp with a heavy spore load impossible to con- trol with Sprays during harvest (12). Botrytis cinerea is able to penetrate unwounded tissue (1). Brown (5) found that penetration of the cuticle must take place in a purely mechanical way, the infecting germ tubes being unable to affect chemically the cuticle of the host nor are they able to secrete any toxic substance which can pass through the cuticle or bring about death to the underlying cells. This pressure theory was backed by Blackmen and Welsfords' (4) work on Vicia_faba. Figure I Egtrztis cinercg, Grey “old fruit rot, on the Premier variety. The advanced stage of this rot is typified by masses of grey conidia as noted on upper portion of berry. ‘3‘ Webb (14) investigrted the effect of hydrogen ion concentration on germination of :Qtrvtis conidia and found that variations in range occurred dependent upon the media. Germination occurred at a pH as low as 1.6 in a nanite solution and as high as pH 10.0 in a beet docoction. In general the eptinun pH for germination was in the area of 3.0. Tribe (13) found hat enzyme preparations from culture filtrates were active from pH 3.5 to 6.0 and that activity decreased rapidly from 6.0 to 8.0. Stevens (11) noted no difference in varietal sus- ceptibility when an epidemic of Botrvtis swept through an experimental plot containing over three hundred vari- eties. Anderson (1) also found no difference between varieties as regards susceptibility to infection by ggtrvtis. However he noted that habit of g‘owth caused a striking difference in the amount of Grey lold which developed in varieties. Varieties which have conra t growth and long leaf stems which shade fruit are more likely to rot. Within the fruit, the fungrs is evidently capable of readily dissolving the Liddle lanella and of pene- trating the cell walls themselves (9). Lyphae grow through the berry rotting the tissue (1) ane the whole berry is rapidly involved, finally becoming covered with conidia (8). The affected berries retain their shape, begin to shrivel but no leakage of juice occurs (9). The berries are even firmer th m: no rnal berries, some eventually becoming hard and dry (1,11). Ehta of Liller and Waggoner (7) collected from a hirst spore trap suggested the most infection by ;otrvtis ciner a origina to es from nearby prinaiy L ioculun and that the micr oelimate afforded by the dense foliage is more import: nt than environmental conditions above the plant in determining incidence of Grey Lbld. Selerotia are produced by the fung us as a protection against dry weather. These lodge in the mulch or drop to the ground and when moisture is present, send out vegetative hyphae which in turn produce conidia (l). Epiphytotics of Grey leld are associated with abund- ant noisture (1,3,17). Wilson (16) in studying the effects of irrigation found that on one occasion when very heavy rainfall followed heavy irrigation and another occasion when hunid at“ sphere prevailed in spite of a diy soil, the increase in crop was offset by an increase in rotten fruit and the marketable crop was slightly less or no more than that from unwztered plots. In culture, growth of Botrytis cinerea at 00C is sparse; at 200 it grows freely; growth at 2500 is lux- uriant and this temperature is consilered in the optimum range for growth falls off snsrply at 3000 (10), Evidence of exploration of variation in the ability of tile fruit of dif1“ ercnt varieties to support Cr<1y hold is apparent137 lacking in the literature. LATERIALS LID {BTHODS l. Prenzretion of otrsubcirv Juices Ripe fruit samples of strswberry selections end varieties were taken at rend m from triel plots located at the C. O. Zollar fiurscry, Benton L. rbor, Lich gen. A puree was then mcde from these berries by means of a laboratory puree urchine, placed in erlenneyer flesks, and stored at 100C. In order to facilitate juice extraction from the purees, one to two cups of moist canntrs cellulose was mixed with each liter of puree before pre,si :. The uice w '3‘.) U) then extracted using a hydraulic press a C.Jo proximately 2500 pounds of pr ssure per square inch. After e} :trection, the individual juices were passed first through a #C3 clarifying pcd on a 60 mm Seitz filter using both Vacuum and air pressure (Fig.2). This was followed by p: ssege through a #ST-3 sterilizing pad and hen final aseptic passage through another #ST—3 sterilizing pad. 2. Solid ledie a. Pet ri Plete IOthd To quickly and accuratelv mersr re t1 e liquid media and ager used in his and following methods, an automatic s;r:1n e was constructed. This consisted of a 20 ml hypodermic syringe inserted through a #13 rubber stepper. A six inch bolt inserted tLrough a rfiigure II Seitz filter and suction flask epprratus Ilsed to clarify and sterilize the strewberry juice. #6 rubber stopper was used as an aejustable plunger stop. This bolt was run through the #13 stopper ad— jacent to the syringe and wes held in place by nuts on both sides of the stepper. A two inch 18 g hypo- dermic needle completed the apparatus Fig.3). Botrvtis CiiPTdRAWES cultured at 200C in petri dishes containing pot to dextrose eger (Difco). Discs, 1.5 cm in diameter, were cut from the peri- meter of the growing culture Lsing a sterile pyrex test tube. Seven ml portions of the sterile strewberry j ice were aseptically pipetted to sterile 9L mi petri plates and mixed with seven ml of cool but fluid ster- ile 3S Difco agar. When solid, the sanples were in— ) oeuleted in the center with discs of Sotrvtip gquwgp, ‘ l I Two metho s of inoculum placement were evaluated in this test. One in which the inoculum wzs plzeed upright and the other in which the inoculum was invert- ed on the agar juice preparation. It was observed that when the inoculum disc wrs inverted and became somet-I} at sealed with the a;_,rr, the I'.‘:.TCCliC growth was delayed slightly in comprrison to pltcing the inoculum disc in an upright manner. The inoculim disc was plrced upright on the agar juice preparation. Inoculeted samples were incubated at 200C and growth measurements taken periodically. Leasurenen s, were made in two directi ns at right angles, eech Figure III Automatic syringe apparatus used to quick y and accurately measure liquid media and agar. measurement taken to the nearest millimeter. In cases where colony growth was irregular, meaSirenents were .1 made along the long a 1 short diameter and the average ‘15 p t:d;-n. b. Growth Tube Lethod The growth tubes consisted of horizontal pyrex glass tubes of a" bore, 15 inches long, bent up 2% inches from each end at a 60° angle. The tubes were held with their ends upright by grooved wooden supports (Fig.4). Using the automatic syringe, six ml of 3p Difco agar was placed in each tube, the tubes plugged, placed in the supports and autoclaved. After sterilization, six ml of sterile strawberry juice was aseptically pipetted into each of the tubes and then each end of the tube alternately raised in a rocking motion to blend the agar and juice. Inoculation of the growth tubes was by means of a small cube cut from a PDA culture of Botrytis cinerea. The tubes were incubated at 200C and growth measure- ments made periodically. ' 3. Lieuid hedia a. Shake Culture hethod The method used for preparing the inoculum for this test was a modification of that described by Nikon, Keller, Sehelling and Stockli (15). Figure IV Pyrex growth tubes showing their linear shape and method of support. A.lO to 15 day old plate culture of Iotrvtis .1 dea to a st rilizcd 1000 nl :- C (‘3 U) r3) cineree on PDA‘w' suction flesx containing about an inch of #6 solid 1 glass beads €31 enough buffer solution, pH 6, to just cover the herds (Fig.5). The flask was swirled until the mycelium was even y brolcen 12p into a fine suspension. One ml was aseptically pipetted into 250 ml flasks containing fift: ml of sterile strawberry juice. T10 inoculated flaslzs were placed on a shaker table for a period of 10 days. The length of the incubation period is greatly dependent upon the fine- ness and density of the nycelie 1 suspension. For comparison between runs it would be necessary to run a standard flash of potato b1 oth or nutrient. After 10 days incubation, the culture was filter- ed through a piece of parachute silk in e buchner funnel. The nycelium was scraped from the silk and placed in a pro-weighed Caluninum dish and dried at 600C to constant weight. b. Spore Germination hethod- Greenhouse Grown Berries -*ore germination tests were made in fresh juice fro: _f*e strawberries taken at random from breeding stoe‘ frawing in the rroenhouse. Juice was expressed by s rennin? the hulled berries on a 6 x 6 inch piece of paper tewcling placed over a 7 x 7 inch piece of cheese cloth. The corners of the cheese cloth were Figure V Shake culture inoculum flask, showing the #6 solid glass beads immersed in the buffer solution. taken up to form a bar and the bag twisted to exp ess juice from the berries. A small drOp of juice was expressed onto the center of a 22 mm seuare cover ma glass and dusted with Lotrvtis spores. lie spores were stirred into the dron with the tip of an inocu- lating needle. The cover glass was hen inverted onto a depression slide previously prepared with a hin layer of vaseline around the edge of the depression. Observations were made after six and nine hours in— cubation at room temperature. After twelve hours in- cubation, germ tube elongetion was too extensive for preper evaluation. The germ tubes of germinated spores were measured at random in units of spore length. For example, a germ tube that was relatively three times longer than its spore was recorded as three. Ten germ tubes were measured at random in each replicate. Where >ossible, a series was run using both two and four berries per juice expression with four slides from each expression. c. Spore Germination hethod- Field Grown Berries Three berries were used per variety and each stage of ripeness. Four slides were prepared for each group and measirenents made on three. Ten germ tubes were measured in each slide using a micrometer eye- piece and low power objective. 4. Whole Fruit Inoculation Tethod Twenty-five fruit of each variety were tested in V-- ‘ u tapes of maturity. Lech the green, white and ripe (I! berry was inoculated on the side with an inverted 3 X 3 mr nycelial cube cut from a 6 to 8 day PDA cul- ture of Botrztis cirerea (Fig.6). hycelial cubes were used as inoculun in preference to spore inoculations because of the ease of locelizing the inoculation and in locating the point of inoculation at the time of evaluation. The inoculated berries were placed on a 13 x 17" glass .late re ting on six small corks in a 14 X 18" D flat. The flat was placed in 3 65°F moist chamber and covered with a plastic covered screen to prevent the U) ist pray from contacting the berries. A temperature of 650E is considered within the Optimum range for growth of Botrvtis while tending to inhibit the growth of rhizonus contaminents. After a four day incubation period evaluations of infection were made by cutting the berry longitudinally through the point of inoculation and neasurin: the depth of visible fuhgal develOpnent. This penetration was ) and was neasured to its 0\ visible as a darkened area (Fi . 8 deepest point and grouped in classes of 5 mm each, from O to 25. Figure VI C e» *“3 m 3 section of inoculated whole fruit after three days incubation. Area of nycelial penetration AAx.’ 1 n ’ ‘ s delined by the brown area on the left p. side of the herry. .J C!“ r ) C’) d- *‘3 l. ' he 3: and Per Cent Soluble Solids of f“) 15.. 12......10 1‘ I": Elli ess. The pH and per cent soluble solids of the various juices were measured following sterilization by Seitz filtration. This data is summarized in Table I. As indicated in Table I the pH varied from 2.6 in selection 2—2 to 3.6 in the Albritton variety. The percentage of soluble solids ranged from 3.2 in the Earlidawn variety to 5.6 in the Albritton variety. Examination of the table revealed no dependent rela— tion between pH and the per cent soluble solids. TABLE I TIE PM" :11) SOLULLE iSOLI33** CF 3311:]. ELL—1211’ U: CE LXIRACTS I Variety pH Solublepsolids 1. 23-2 2.6 4.8 2. Enpire 2.7 4.3 3. South Haven 195 2.7 #.2 h. Z-l 2.8 #.7 5. South Haven 44 2.9 .8 6. Eerlidewn 3.0 3.2 7. Sparkle 3.1 3. 8. Red Crop 3.1 #3 9. Robinson 3.2 4.7 O. Fairlend 3.2 3-3 1. Premier (flows-rd 17) 3.2 4.2 2. Albritton .6 5.6 * Becknan glass electrode ijmeter Lodel hé2 ** Bausch and Lomb hand model refractoneter 2. P C) tri Plate, C—roz-zth Tube and Slel:e ilture *3 n C ;ble II contains a summary of date obtained on he effect of strrwoei ry juice incorrorzted in solid and liquid media in petri plates, re th tubes and shake cultures upon the growth of ;btr tis cinerce This permits a comparison of results obtained by the tnree F'h methods. no ret— CI.) of growth of iotrytis cineze; on Albritton using the petri pl: te method, is twice thrt which occurred on Premier (Howcrd l7), nearly three times that oi Iobinson and four times the rate that occurred on the Z-2 selections. The order of results obtained by the growth tube 1 method ere si’ iler to tie e o»tained hy the petri plrte - metlod as shown in the same table. The Robinson 01o lbrlidewn varieties indicated good growth while the iner c On the H'— Empiro variety failed to support a. verieties Fairlend, Empire and South lb ven 195 w} ere no growth occurred, inoculrtions were repeated several times, each failing to grow. In the shelze culture test, juice from Albritton gain wzs nest feverrble for growth of Betrytis cinc1ee , b 1ollowed in order by Premier (Hewrrd l7), Iooi:1 son and Eerlidewn, which is comparable to the results obtained in the Petri Plate method. ~firthcifiore, these results in this test subs t: ntiete tiose obtained for :‘Lobinson end Berlioewn in growth tubes. Also, Z-l, hhpire, TABLE II TEE L“;LCT OF QELIJ"LLL” JUICL I; SOLID L;D LL LID- -LLIA r~~ v T r‘1' ' "r‘ '. T‘. ."~7" “"1 " v -\ I'v-‘A- J~1rv71 ‘ ~ . II«C OILI 014%..qu .I.-'- I'ILJIIL: III). L..J..JS, u‘- LUNIII .LL.'-->...'n:) .LlLI-U QILI'II-IE) (I‘79T‘ ',|" I] 715"] 7 071‘ . "Tin-"1:31” ”“ ‘7,"’v'_""1h GUI—J-.. UILLJ 0;: .LA I.) GIL 2.4;..- J.‘ LJ _LI‘L 0 CS... I.)IL..).{1 Aversge Daily Incrcrso in_§rowth Shake Potri Elete Growth Eube Culture Diemet er Length ugt. gns. Variety 1L1 Lul l. Albritton 35.0 -* .46 2. Premier (Howard 17) 15.6 - O U) \n U.) 3. Robinson 12.6 0 O\ O o O\ O CO 4. Ibrlidawn 12.0 5. Red Crop 12.0 - . Fairland 9.8 O 6 7- Z-2 8.0 — 8 9. Eipire O O 10. South Levon 44 O - ll. South he ven 195 -» O l O O O O O O O O O H 12. Sparkle — 6.6 * Variety not tested P.) 1..) South Levon uh and South Haven 195 are similar between methods in that each failed to support ;. cincrep in ither method. Ibwevcr, there is a discrepancy, in that Red Crop, Fairlsnd end Z-2, supported Botrvtis c n3r a in the petri plate method while Sparkle support- ed Botrvtis in on y the growth tube method. In the shake culture test, all three varieties, Wed Crop, Fairland and Z—2, failed to support the growth of Eotrvtis cinerea. This may be due to a difference in the sen- sitivity of the two methods or it could possibly_be due to the dilution of the juices by the agar when using the plate and growth tube methods. 3. Snore Germination a. Greenhouse Grown Berries rhe results of snore germination studies on juice of greenhouse grown berries is presented in Table III. This method consisted of illOCUl tions of a hen.;ing d1 op preparation of strswberry juice with 2. cireree spores. Germ tube long ths are in units of spore length. As given in Table III, juice from selection South Javen #58 is most I: vorable for go rm tube growth fol- lowed by South Haven l9h-, ELrlidawn, South H-ven 295 and Ehst Laflhsg1065. There is no significant dif— ference between a two or a four berry sample. The results obtained are due to varietal influence rather then srmple size. TABLE III SPORE GERLILATIOE OE QTJIT'“ = ILEEiA IE LILAJJJRHY JUICLS 11L “AT“ 4".) F110;- CELELTE LSE GEOJLI ELJZILS 30- 0f 10- Of Rolrtivc Corn “The Lcn£:h* Selection berries Reps. Incu:r ion rcriod 6 lIl’S. 9 4‘TSQ 1. Check “lass - M 0 0 Diet 1120) 1.8 4.2 m 0 t5: 0 L" o *‘T’ C) O \51 IX) .C' 4 2.3 6.4 1.8 5.8 n) \C) * \n \n F‘ -F 10 g 4 5.5 10.2 \n O c: 0 : [—4 \o ¥i P0 H \ 3 - .3. r— .l- q 1 6.4 10.5 * iclrtive length of germ tlbc i11 units of 2“ore lcncth Selection L.S.D. .05 = 3.2 Sel. X Inc. Period L.S.D. .05 : 2.3; .01 : 4.1 23 I With exception of nerlittri, the verietihs test- ed in tLis net od ere not incluete in the methods in Ttble II, thcre?orc, 0 true correlrtion cannot ha mete between the two tables. Lowever, Lerlidewn re- mains favorehle for the growth of botrvtis in the spore germination nethod es in the previous nethoas (Table II). b. Field Grown berries (I) L) x - ' .. -F. n .,.. : J--- ,_ :7 :- _, . n ., - ...- inc in:flue;ce oi Vtriet; «flu SbrfiC oi lipeLe 't‘," 1"- U ‘ ‘4‘1fi ‘r‘ P " -1\v"‘ 4“) I “ (V p _ "I ‘5‘ fl" “lfi 1 A T‘ u on. ;ic (,-ogtnr CI £52,“. tutw.s 01 :i- c u;c-t1 tueicz ‘l I W o 1 _o _ ‘ stueiee in this test. ve of the eL ht selections PJ- As given in Table IV, f under test were more frvor hle for germ tube growtl H. :_ 1‘ ~ I. J-fi 0- fln ‘ v-v -' (\‘L‘ ‘-).J' . ’L -‘ ". the 331 to Sbv$e oi UCTTJ flvuhllby then in th ripe trfe. Verietel differences were else eviflenced. U) P Albritton JCS tLe Lost favorable ior fern tube develop- ncnt, enfl South Lrvcn 195, the lcrst favorable. Compsring with Table III, sclectiens South Irvcn 2? and Last Lansing 1365 maintained sinilrr ranks in Table IV as in Teole III, while selection South Haven 194 hrs shifted from the more unfavorable in Table III to the more favorable for g rozth in I? ale IV. 4. Growth of Isolrtes of Lotrjtis cineree into 3033?]. ' S q A study was meoe 13ing whole strawberry fruit as the substrete for botrytis cinere e in order to determine if the varietal relationshL,s iouzid in the juice TABLE IV GEEXILATIOI OF ZOTLZTIS CILELEA SIORES AFTER SIX ”0' ILCDBATIUH IL JLICE OF D TELLLLE TARILT I'S 03 FIELD GROI‘JZI 81 WE .LJELEZLIES AT Z'ILIITE AL.) LIFE STAB 8 OF BERRY LAbeift 1* (mean) Aversge Germ Tube Lenr; Variety Jhite Iupe 10 8. II. 195 S. L. 109+ Premier (Howard 17) 11.4 12.2 2.1 10.1 12.0 111.1 0 Fairlfnd 150 13.6 11+.5 lbbinson 14.3 16.9 \J O\ \n ¥' 0 E. L. 1065 S. H. 295' P) H l-’ \0 O I C\ C'\ LU }_-J C\ O C m 1C.0 15.0 8. Albritton 19.5 21.0 20.0 Averag 16.0 1#.9 * Average of 30 spores in units of eyeoiece micrometer, Varieties = L.J.J. lwqturitics =" 1.103 01). low power 'V’ V x L groups): objective. .05 = .05 :2 0.5; 001 "CL Sol). 005 =‘108; 4 ,rep rations could also be noted in the whole berry, and to determine if these relationshins exist at the - various stress in the development of the fruit. As indicated in Table V, degree of penetration of the whole fruit increased with increased maturity with the exception of selection East Lansing 1065. however, W the degree of incr see in penetration was dependent upon variety. For example, Prenier (Howard 17) had an average penetration in the green and ripe stages of 9.1 and 16.5 nu respective y. A difference of 7.4 mm. Whereas, selection South Haven 295 had an evercge pene- tration in the green and ripe stages of 8.3 and 29.5 mm respectively, a difference of 14.2 mm. This dependence of penetration upon variety is further evidenced by the significant interaction between variety and ripe- The difference between varieties and between stages of ripeness and the interaction between them were highly significant. It is suggested that the variety reaction to Zetrytis cinerea infection depends upon stage of nat- urity as well as variety and one cannot predict a re- ction without takine both factors into consideration. {D Correlation coefficients were calculated with data obtained in the spore germination studies on field grown berries (Table V) and penetration studies of whole fruits (Table V) as well as for different stages of ripeness within each group. Correlation coefficients are summariz- TALJLE V 6‘a Hero f1 1 M1 “3‘7“." ‘11? 0111021..) 01‘ 1~.1»11L=1..L.L_1- 111. 1; Selection Green 1. E. L. lfo5 4.0 2. S. K. 105 4.0 4 3. s. 1:. 19 5.9 4. Premier 9.1 (row-rd 17) 5. Robinson OI‘EKLOIJS STIJEJJLLJIK , r1 7,1771 Cinni 1£1 it e 11.8 10.0 10.5 16.1 Stag of Fruit Development (neans' q Ripe 1.2 7-9 14.5 16.5 16.9 17.5 22.5 J Avercge 10.4 * heasurenent in mm. Selection - L.S.D. liturity — L.S.D. .05 = 0.6; 0 V x h (group) - L.S.D. 13.9 . —. .¢-- — -.~ .1 -. _. ,. - A, c- in 1aele VI. The reaction of selection Last Lansing 136; to nycclial penetration in rize fruit was not in line with its rerction in other stares or other tests . 1his is particularly evident by the negative correlation coef— ficient obtained between the length of fiern tubes in juice of white berries ant the nycelial leneer1tion pf whole ripe fruit. 1H8 cause is Linnown and ray perhaps be due to a 1ode of resistrnce peculiar to this selection. Further testing of other selections will per11 cps reveal the cause or at least reveal whatner other selections will follow this sane pattern. For purpose of corre- lation between Tables IV axe V, two correla ion coeffi- ciezits were run, includ in” and :xclic'n" selection tht Lansing 1065. Correlation tests includi1g selection Les t L'“°l£§ 1065 t)ave a hijh y significant correlation coefficient of .97 between the growth of gore: tubes in juice of ripe fruit and nycelial penetration of fllOlG white fruit. Also, nycelial penetration of whole white fruit and nycelial penetration of whole ripe fruit gave.a signi- ficant correlation coefficient of .70. All other corre- gnif icant. p. lation coefficients obtained were non- Correlation tests excluding selection Last La nsing rave a highly sign1ificant correlation cocfiicient of .9H between germ tube growth in juice of ripe fruit TALLL VI TLL'LS OF CULRLLLTIOL HITLIH AID ILTVLLT GLLL 1ULL STLDILS (TALLL IV) ALD NIOLL LLLL: 1L.L1LL1IU LTLU1-L ( 1LLLL f) Ziethod COClli: c Germ Tube White vs Penetrrtion White .L6 .26 Germ Tube Ripe vs Penetretion Ripe .9%** .5 Geni Tube Riie vs Penetration White .99** ~97** Gena Tube Pnite vs Penetretion Line .63 ~.l§ Germ Tube White vs Germ Tube Ripe .54 .46 Penetretion White vs Penetration Ripe .99** .70* Penetretion Green vs Penetration Ripe .69 .30 Penetration Green vs Penetration White .52 .50 A. Selections - South Haven 195, 194, 295, Feirlsnd, Prenier (Lewerd l7) enl Robinson. 3. Selections -Eest Lens1n 1065, Sei1th Lav n l,,, 194, 295, Fairlend, Premier (ibwerd l7) and Robinson. ** .01 level of si1nificence * .05 level of significance 20 and nyceliel penetration of whole ripe frui . lighly a - 1 an, _ .96111ere lettlA- H- [—1 H. O H. I '3 C+ 3 0 P1“. ‘7 i“ k ._..-.J_.L. cent correlction coex ed between germ tube erowth in juice of ripe fruit rnL penetrrtion of whole rine fruit es well es ' \‘L ‘ . 'q ‘3 ‘ 4 D . r. " L V‘*' . r. .enetretion 01 whole wxlte 1ru1t Ln: chelicl penetrrtion of whole rine fruit. All other correlation 4. coefficients obtrinefi were non-signi1icent. Dl.fl3LSS ICES Differences in the ability of the extrrcted juice of vsrious strewberry verirtir to euphort fetrvtis cineree were 21*erent on esr' and lieuir 1 - ' ”0’" ““"‘\r‘ "1. . 43 . by spore jernin1t1on in juice. The petri plate method in€iceted thrt juice of the varieties Albritton, Frenier (Iowerd l7) and Lobinson ,4 venwal1i ghly fevcnruile for the :1T1m1}. of lotr W1: 3 ciwerec. The entire plrtes were covered within six days, whereas preu151‘etions 01 Feirlrnd end 2-2 verietibs hrd not shown any increese until the fourth or fifth deys re- I Spectively end no growth was visible :1 ell on .L.1,.| ”3. L110 4.1- pire, South haven #4 end the Z—l prejeretions. The order of results out: ined in the ;;ro th t1oe method were similar to those ooteined by the petri plate method with the execution of the :eirlrnd veriety ULiCJ isol ye growth in the pctri ulrte but faileC to sup- port lkfinrttis cineree i11'miis method, The resu ts in licu1ie media for Albritton, Premier (Howcrd l7), Ibbinson end Errlidewn were si11iler to tH1o e obtained in solid media. However, Red Crop, Fair and ene Z-2 varieties which supported Dotrrtis ci1