MANAGING ANTHRACNOSE BLIGHT AND BOTRYOSPHAERIA AND PHOMOPSIS CANKERS OF WALNUT PART 1: BOTRYOSPHAERIACEAE AND PHOMOPSIS CANKERS OF WALNUT

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1 MANAGING ANTHRACNOSE BLIGHT AND BOTRYOSPHAERIA AND PHOMOPSIS CANKERS OF WALNUT PART 1: BOTRYOSPHAERIACEAE AND PHOMOPSIS CANKERS OF WALNUT Themis J. Michilides, Shuifei Chen, Bill Cotes, Dvid Morgn, Ryn Puckett, Jnine Hsey, Kthy Anderson, Richrd Buchner, Crolyn DeBuse, Elizeth Fichtner, nd Wlter Bentley ABSTRACT A totl of 185 isoltes of Botryosphericee nd Phomopsis spp. were collected from 13 counties in Cliforni such s Butte, Colus, Fresno, Kings, Glenn, Merced, Sn Benito, Stnislus, Sutter, Tulre, Ventur, Yolo, nd Yu. Identifiction of the isoltes ws sed on morphologicl fetures of the cultures nd DNA sequence comprisons of ITS, TEF-1α, nd β-tuulin gene regions, 1 species of Botryosphericee nd two species of Diporthe (nmorph Phomopsis, Diporthcee) were identified. These species include Botryospheri dothide, Diplodi serit, Diplodi mutil, Dothiorell ieric, Lsiodiplodi citricol, Neofusicoccum mediterrneum, Neofusicoccum nonquesitum, Neofusicoccum prvum, Neofusicoccum vitifusiforme, Neoscytlidium dimiditum, Diporthe rhusicol nd Diporthe neotheicol. Neofusicoccum mediterrneum ws widely distriuted in 1 of the 13 smpled counties. Pthogenicity on three wlnut cultivrs, Chndler, Tulre, nd Vin, using mycelil-plug inocultion method showed tht ll these species re potentil pthogens to ll the tested wlnut cultivrs, with L. citricol, N. prvum eing the most pthogenic species, followed y N. mediterrneum, B. dothide nd N. dimiditum. The cultivr Chndler ws more tolernt to infection thn those of Tulre nd Vin cultivrs. The virulence of Botryosphericee to wlnut decresed from summer to the fll. Liquid extrcted from the wlnut hulls did not ffect the mycelil growth of the Botryosphericee species. However, this liquid inhiited significntly the spore germintion of Botryosphericee isoltes. With the progression of the seson from summer to fll, the volume of wlnut fruit hull extrct decresed s did its effect on the germintion of conidi of Botryosphericee spp. Wlnut rnches infested with scle collected from the field nd inoculted with three Botryosphericee fungi were more susceptile to cnker formtion thn the rnches without scle. OBJECTIVES 1) Identify the cnker pthogens on wlnut smples from frm dvisors nd growers, s well s from our surveys, clrify the distriution of these pthogens in the wlnut growing res in Cliforni. 2) Determine nd compre pthogenicity of the vrious fungl isoltes nd fctors ffecting infection of wlnut. 3) Determine puttive ssocition of wlnut scle nd infection y these fungl pthogens. Cliforni Wlnut Bord 367 Wlnut Reserch Reports 12

2 PROCEDURES 1) Identifiction nd distriution of Botryosphericee isoltes A totl of 185 fungl isoltes from wlnut showing typicl morphology of Botryosphericee or Diporthe fungi were trnsferred from our culture collection. The isoltes were collected from disesed wlnut smples originted from 13 counties of Cliforni (Butte, Colus, Fresno, Kings, Glenn, Merced, Sn Benito, Stnislus, Sutter, Tulre, Ventur, Yolo, nd Yu). Identifiction of the isoltes ws sed on culture morphologicl chrcteristics nd DNA sequence comprisons of ITS, TEF-1α, nd β-tuulin gene regions. 2) Pthogenicity tests To test pthogenicity, one to three isoltes of ech identified species were selected for the field inocultion tests. Three cultivrs, Chndler, Tulre, nd Vin of Juglns regi were selected for inocultions. Pthogenicity tests were conducted y inoculting two-yer-old rnches nd the current hulls of the fruit. Inocultions were conducted y using 7-mm mycelium PDA plug from 7-dy-old culture. Wounding of the rk nd hulls ws creted with 7-mm cork orer, nd plcing n gr plug ering myceli, upper surfce down into the fresh wound. Five two-yer-old rnches or hulls of five fruit per isolte were inoculted. Five dditionl rnches or hulls of ech of the three cultivrs were wounded nd inoculted with sterile PDA plug nd served s negtive controls. Two experiments (the first in July nd the second in August) were conducted using the sme methodology. Infection dt were recorded 3 weeks fter inocultion y mesuring length of rnch cnker nd fruit light. In this study, results of experiments were nlyzed with SAS (version 9.3, 11) using the PROC generl liner model (GLM). Anlysis of vrince (ANOVA) ws performed on the dt nd tretment mens were compred using Tukey s LSD test t P =.5. 2) Effect of inocultion method nd seson on infection of wlnut Two isoltes (5H53 nd 5C87) of N. mediterrneum, one isolte of N. prvum (isolte 5B6), one isolte of B. dothide (isolte 2E55), nd one isolte of L. citricol (isolte 6I35) were selected for this study. Brnches nd fruit on wlnut trees were inoculted either with the mycelil plug or the conidil suspension inocultion method. The mycelil plug inocultion method ws performed s performed ove. For the conidil suspension inocultion, suspension of conidi per ml ws used. Pthogenicity tests were conducted y inoculting five two-yer-old rnches or hulls of five fruits per isolte for ech inocultion method. Five dditionl rnches or hulls of ech of the three cultivrs were inoculted with sterile PDA plug or wter nd served s negtive controls. All the inocultions were conducted on the three wlnut cultivrs Chndler, Tulre nd Vin. Five inocultion experiments were conducted from July to Oct. 12. After three weeks of ech inocultion, the results on the rnches nd fruits were recorded on Aug. 13, Sept. 11, Oct. 1, Oct. 22, nd Nov. 13, respectively. Cliforni Wlnut Bord 368 Wlnut Reserch Reports 12

3 2) Effect of hull extrct on mycelil growth nd spore germintion In order to test whether the hull extrct hs ny effect on the growth of isoltes of Botryosphericee nd on the infection rtes of the vrious species of the Botryosphericee, the following experiments were conducted. Helthy wlnuts from the sme rnch of the sme tree were collected in five different times, i.e., on Aug. 25, Sept. 15, Sept. 3, Oct. 15, nd Oct. 3. from ech of the three wlnut cultivrs Chndler, Tulre nd Vin. The hulls of these wlnuts were seprted, cm 3 of hulls ground with mortr nd pestle, in the liquid extrct ws collected nd mesured. To test the effect of hull extrct on mycelil growth, 7-mm mycelium PDA plug from 7-dyold culture of ech isolte tested ws trnsferred to pltes with wter gr (WA, 1 g gr, 5 ml wter) nd 1 µl of the extrct ws plced on top of the mycelium plug; mycelil plugs received 1 µl of wter served s controls. Four fungl isoltes (5C87 of N. mediterrneum, 5B6 of N. prvum, 2E55 of B. dothide, nd 6I35 of L. citricol) were used in this experiment. Five pltes with WA medi per isolte nd control nd for ech cultivr were tested. Mycelil growth ws recorded y mesuring the dimeter of the culture colony fter 72 hours incution t 25 C. To test the effects of the hull extrct on spore germintion, conidil suspension ( conidi per ml) ws mixed with vrious diluted solutions of the extrct, such s 1%, 5%, 25%, 12.5%, 6.25%, 3.13%, 1.56%,.78%,.39%,.%, nd.1% extrct. An liquot of µl of the spore-extrct mixture ws plced WA medi, nd the pltes were incuted t 25 C for 8 hours. Conidi suspensions of four fungl isoltes (5C87, 5B6, 2E55 nd 6I35) were used. µl of conidi suspension in wter (without the hull extrct) per isolte plced on WA pltes served s controls. The 5% vlues of the germintion of conidi were clculted. 3) Puttive ssocition of wlnut scle nd infection y these fungl pthogens To determine whether wlnut scle predisposes wlnut rnches to infection y the Botryosphericee fungi, one isolte from ech L. citricol (isolte 6I35), N. prvum (isolte 7E81), nd N. mediterrneum (isolte 5C87) ws selected. For ech isolte, 5 2-yer-old rnches infested y wlnut scles nd 5 2-yer-old rnches without scle were collected from the cv. Vin nd used for this experiment. A conidil suspension of conidi per ml ws prepred nd µl of the spore suspension ws plced on pre-mrked re on ech of the rnches (those with scle nd the ones without scle). For control rnches (with scle nd without scle), µl of sterile wter ws plced similrly. All the rnches were incuted under 1% reltive humidity in plstic continers t 25 C. The experiment ws conducted on 6 Oct. 12, the incidence (%) infected rnches ws determined fter 3 weeks. To determine whether the wlnut rnches with scles re more susceptile to Botryosphericee thn the rnches without scle, isoltes 6I3, 7E81 nd 5C87, were lso inoculted on two-yer-old cultivr Vin rnches with scles nd without scle, respectively. A 7-mm mycelium PDA plug from 7-dy-old culture ws used for the inocultion. For ech isolte, 5 rnches with wlnut scles nd 5 rnches without scles were inoculted. Brnches were inoculted with sterile PDA plug served s negtive controls. All the rnches were cultivted in the 1% humid plstic continers in 25 C. The experiment ws conducted on Oct. 6, 12; the lesion length produced y the mycelil plugs ws mesured fter one week. Cliforni Wlnut Bord 369 Wlnut Reserch Reports 12

4 RESULTS AND DISCUSSION 1) Identifiction nd distriution Using sequence dt of three genes (ITS, TEF-1α, nd β-tuulin), the 185 isoltes collected from disesed wlnuts in Cliforni elonged to 1 different species of Botryosphericee. These include Botryospheri dothide, Diplodi serit, Diplodi mutil, Dothiorell ieric, Lsiodiplodi citricol, Neofusicoccum mediterrneum, Neofusicoccum nonquesitum, Neofusicoccum prvum, Neofusicoccum vitifusiforme, nd Neoscytlidium dimiditum. In ddition, two species of Diporthe (Diporthe rhusicol nd Diporthe neotheicol tht elong in the Diporthcee) were recovered from infected wlnut (Tle 1). Neofusicoccum mediterrneum is dominnt, 138 of the 185 (75%) of isoltes were identified s this fungus. Neofusicoccum mediterrneum ws widely distriuted nd ws recovered from 1 different counties (Tle 1). In ddition to Cliforni, two isoltes from wlnuts from Greece were identified s B. dothide nd mong nine wlnut isoltes from Spin eight were identified s N. prvum nd one D. mutil (Tle 2). Botryospheri light hs cused significnt yield losses in wlnuts grown in Spin (T. Michilides, personl communiction) nd the disese hs een reported in wlnuts grown in Greece. 2) Pthogenicity tests A totl of 21 isoltes, representing the 1 species of Botryosphericee nd the two Diporthe spp. were selected for pthogenicity tests (Tles 3 & 4). The verge lesion length for the comintion of two experiments is shown in Tles 3 & 4 nd Figures 1 & 2. On ttched rnches, ll the tested fungi were le to produce longer lesion on the three tested cultivrs thn the non inoculted control rnches (Tle 3). All the tested isoltes of L. citricol nd N. prvum produced significntly longer lesion thn control (Tle 3). Except isolte 5H9 of N. mediterrneum on Chndler, ll the tested isoltes of N. mediterrneum produced significntly longer lesions thn the control in ll three cultivrs (Tles 3). When results of the three cultivrs were comined, clerly L. citricol nd N. prvum were the most pthogenic species, followed y N. mediterrneum, N. dimiditum, B. dothide nd N. nonquesitum (Figure 1). Isoltes of L. theorome nd N. prvum were le to kill the ttched rnches in ll three test cultivrs. Isoltes of N. mediterrneum killed the ttched rnches of Tulre nd Vin in 3 weeks fter inocultion. The results from inocultions of fruit hulls on the trees were consistent with those from rnch inocultions. All the tested fungi produced wider decy re thn tht in the control fruit (Tle 4). With the exception of D. serit, D. mutil, D. ieric, D. rhusicol nd D. neotheicol, isoltes of ll the other species produced significntly lrger lesion on hulls of the three cultivrs (Tle 4). After comining the results for the three cultivrs, N. prvum nd L. citricol were the most pthogenic, followed y B. dothide, N. mediterrneum, N. dimiditum nd N. nonquesitum (Figure 2). For cvs. Tulre nd Vin, ll the species of Botryosphericee were le to rot the entire hull of the fruits in 3 weeks. However, on cv. Chndler, D. serit, D. mutil nd D. ieric, decyed only prt of the hulls. Inocultions with the Diporthe species sowed tht only D. neotheicol ws le to decy the entire fruit hull of cultivr Vin in 3 weeks (Tle 4). The overll results sed on the rnch cnker length nd hull rot dimeter showed tht Chndler is less susceptile to the Botryosphericee nd Diporthe pthogens thn the cvs. Tulre nd Vin (Figures 3 & 4). Cliforni Wlnut Bord 37 Wlnut Reserch Reports 12

5 2) Effect of inocultion method nd seson on infection of wlnut The mycelil plug inocultions showed tht ll the five tested isoltes (Tle 5) produced disesed lesions on the ttched rnches nd decyed the fruit hulls, ut no lesions developed in the control rnches/hulls (Tle 5). However when spore suspension ws used to inoculte rnches nd hulls, only L. citricol resulted in cnker development nd decyed the wlnut hulls (Tle 5). The results suggest tht germintion of spores is importnt for infection of wlnuts nd the conidi of the vrious species of Botryosphericee will differ in their ility to infect wlnuts. It is therefore criticl to determine whether there re compounds in wlnut tissues tht fvour or inhiit the germintion of Botryosphericee conidi on the surfce of wlnut tissues. Periodic inocultions of ttched rnches with mycelil plugs indicted tht the most of the tested fungi produced the longest lesions in the Septemer inocultions nd the shortest lesions were produced in Novemer (Figure 5). Inocultions with mycelil plug on the ttched fruit showed tht ll the tested fungi decyed the entire hulls in 3 weeks. Decy development ws smller with the August inocultion ut incresed significntly s the nuts mtured in Septemer, Octoer, nd Novemer. There were no differences mong the decy developed from Sept. nd Oct. inocultions (Figure 6). For the conidil suspension inocultions, only isolte of L. citricol produced disesed lesion on rnches nd hulls. Wlnut rnches nd fruit hulls were more susceptile to infection (lrge lesion size) during Septemer nd erly Octoer nd less susceptile in August nd lte Oct. (Figures 7 & 8). 2) Effect of hull extrct on mycelil growth nd spore germintion To determine the differences in susceptiility of wlnut hulls to infection y the Botryosphericee fungi, we exmined the mounts of hull extrct otined from hulls s the seson progressed. The volume of liquid extrct decresed from summer to the fll (Figure 9). The growth of the myceli of Botryosphericee isoltes ws not ffected significntly y the wlnut extrct (dt not shown). More thn 9% of the conidi in the wter suspension germinted within 4 hours for ll the four tested isoltes. The wlnut liquid extrct inhiited the germintion significntly of ll four tested isoltes (results only for N. mediterrneum re shown, Figure 1). The inhiition of conidi germintion of Botryosphericee y the wlnut hull extrct decresed from summer to fll (Figure 1). For exmple, to get 5% inhiition of germintion higher concentrtion of the hull extrct ws needed lter in the seson thn erlier in the seson (exmple:.2 to.4% of hull extrct ws necessry for 5% inhiition on 25 Aug. vs..8 to 1.55% on Oct. 15; Figure 1). 3) Determine puttive ssocition of wlnut scle nd infection y these fungl pthogens For ech of the three isoltes, the percentge of rnches infected y the Botryosphericee ws higher for rnches with scle thn tht for rnches with no scle (Figure 11). For exmple, 18% of the rnches with scle were infected y L. citricol while only 6% of the rnches with no scle. These results indicte tht the wlnut rnches with scle somehow re predisposed to fungl infection. More isoltes of the Botryosphericee isolted from wlnut will e needed to e tested in order to mke more generl conclusions on the vrious species of Botryosphericee isolted from wlnut. Cliforni Wlnut Bord 371 Wlnut Reserch Reports 12

6 The mycelil plug inocultion resulted in longer cnkers on rnches with scle thn those without scle infesttion for ech of the three Botryosphericee species used in these experiments (Figure 12). The results suggest tht the tissues of wlnut rnches infested y scle somehow ecome more susceptile to infection y Botryosphericee fungi thn the tissues of rnches without scle. In previous reserch it ws found tht necrotic islnds of tissues tht develop under the feeding sites of the scle re frequently colonized y Botryosphericee nd Diporthe fungi (T. Michilides, personl oservtion). CONCLUSIONS Severl species of Botryosphericee nd Diporthe (Phomopsis spp.) re widely distriuted on vrious cultivrs of wlnuts in numer of counties where wlnuts re grown in Cliforni. Pthogenicity tests indicted tht ll these fungi re pthogenetic to t lest three cultivrs of the English wlnut. Lsiodiplodi citricol nd Neofusicoccum prvum re the most pthogenic, lthough they re not s spred s some of the less virulent species. However, it is needed tht the frequency of these fungi is monitored to determine their spred to other regions nd possile uilt-up of their inoculum in wlnuts. Neofusicoccum mediterrneum, which exhiits moderte virulence, ws found to e the most widely distriuted species in wlnuts. More reserch is needed to determine the mgnitude of thret this pthogen nd the other species of Botryosphericee could pose in wlnuts nd to develop efficient wys to mnge these pthogens. At lest three wlnut cultivrs tested showed susceptiility to infection y the Botryosphericee fungi with Chndler eing less susceptile thn Vin nd Tulre. Initil experiments showed tht species of scle occurring on wlnuts predisposed rnches to infection y Botryosphericee fungi. However, more reserch is needed to determine whether scle +Botryosphericee species would ccelerte the killing of wlnut rnches. Tle 1. Identifiction nd distriution of Botryosphericee nd Diporthe on wlnut in Cliforni. Species No. Species nme No. of isolte Distriution/county 1 Botryospheri dothide 4 Butte Glenn Yu 2 Diplodi serit 13 Fresno Kings Merced Sn Benito Tulre 3 Diplodi mutil 6 Tulre Ventur Yolo 4 Dothiorell ieric 1 Yolo 5 Lsiodiplodi citricol 2 Stnislus 6 Neofusicoccum mediterrneum 138 Butte Colus Fresno Glenn Merced Stnislus Sutter Tulre Yolo Yu 7 Neofusicoccum nonquesitum 4 Colus Sutter Yu 8 Neofusicoccum prvum 6 Butte Stnislus Yolo 9 Neofusicoccum vitifusiforme 3 Fresno 1 Neoscytlidium dimiditum 1 Kings 11 Diporthe rhusicol 6 Kings Stnislus 12 Diporthe neotheicol 1 Stnislus *First report on wlnut in Cliforni. Cliforni Wlnut Bord 372 Wlnut Reserch Reports 12

7 Tle 2. Identifiction nd distriution of Botryosphericee nd Diporthe on wlnut in Greece nd Spin. Country Species No. Species nme No. of isolte Greece 1 Botryospheri dothide* 2 Spin 1 Neofusicoccum prvum* 8 Spin 2 Diplodi mutil* 1 *First report on wlnut in the reltive country. Tle 3. Averge cnker length on rnches of Juglns regi cvs. Chndler, Tulre nd Vin cused y mycelil plug inocultions with Botryosphericee nd Diporthe fungi. Species Isolte No. Cnker length (mm) Chndler Tulre Vin Botryospheri dothide 2E55 32 k-u* 34 k-u 3 l-u 2D 24.2 m-u 37 j-t 58.5 i-k 5A m-u 43.9 j-o 3 l-u Diplodi serit 3H n-u n-u 17.8 n-u 5F n-u 19.9 n-u 18 n-u Diplodi mutil 6F65 22 m-u 16.9 p-u n-u 6B q-u 19.8 n-u n-u Dothiorell ieric 5G97 15 q-u 15.2 q-u 18 n-u Lsiodiplodi citricol 6I35 86 gh cd 196 6I c c 177 Neofusicoccum mediterrneum 5H53 4 j-r 43.3 j-p 37.5 j-s 5H9 32 k-u 48 j-m 37 j-t 5C87 41 j-q 83 g-i 53 j-l Neofusicoccum nonquesitum 5A4 43 j-p 27.2 l-u 26 l-u Neofusicoccum prvum 1L g-i 82 g-i 177 5B62 99 fg 13.5 c-e 14 e-g 5B d-f c Neofusicoccum vitifusiforme 5H q-u 25 m-u 19 n-u Neoscytlidium dimiditum 2D j-n 3.3 l-u 63.5 h-j Diporthe rhusicol 6I q-u 16.5 p-u 15.5 q-u Diporthe neotheicol 6I r-u 16.2 p-u 15.5 q-u Control 9.9 t-u 1.3 s-u 9 u * Numers followed y different letters re significntly different (P =.5). Cliforni Wlnut Bord 373 Wlnut Reserch Reports 12

8 Tle 4. Averge rot dimeter of fruit hulls of Juglns regi cvs. Chndler, Tulre nd Vin cused y mycelil plug inocultions with Botryosphericee nd Diporthe fungi. Species Isolte No. Hull rot dimeter (mm) Chndler Tulre Vin Botryospheri dothide 2E j-o* 52. -f 52 -f 2D 36.6 j-p 44.7 e-j A k-p 44.4 f-j c Diplodi serit 3H t-v 29.7 n-r 41.7 g-l 5F v-x 33.6 l-q 39.8 h-m Diplodi mutil 6F u-w 25 r-u 4.6 h-l 6B v-x 39.3 i-m 42.5 g-k Dothiorell ieric 5G97 13 v-x 21 s-v 26 q-t Lsiodiplodi citricol 6I d I e 6 Neofusicoccum mediterrneum 5H53 4 h-l d 38.5 i-m 5H j-o 39. i-m 41.5 h-l 5C87 41 h-l c 5 -g Neofusicoccum nonquesitum 5A p-s 41.5 h-l 38 i-n Neofusicoccum prvum 1L d 48 c-h 6 5B B Neofusicoccum vitifusiforme 5H2.5 s-v 31.1 o-r 37.5 j-o Neoscytlidium dimiditum 2D57 38 i-n 38 j-o 46 d-n Diporthe rhusicol 6I v-x 15.5 v-x.5 s-v Diporthe neotheicol 6I v-x 16 v-x 31.5 m-r Control 9.9 w-x 9.6 w-x 9 x * Numers followed y different letters re significntly different (P =.5). Tle 5. Lesion lengths produced y Botryosphericee using two different inocultion methods; results show the verge of three cultivrs for ll periodic inocultions of rnches nd fruit hulls on trees. Tretment Species Isolte/No Mycelil plug on rnches Mycelil plug on hulls Conidi suspension on rnches N. mediterrneum 5H d* 52.3 c c 12.9 cd N. mediterrneum 5C c c N. prvum 5B c B. dothide 2E d c c cd L. citricol 6I Control 9.57 e 9.77 d 12.8 c d * Numers followed y different letters re significntly different (P =.5). Conidi suspension on hulls Cliforni Wlnut Bord 374 Wlnut Reserch Reports 12

9 Averge lesion length (mm c c 4 cd de ef ef f f f f Lsiodiplodi citricol Neofusicoccum prvum Neoscytlidium dimiditum Neofusicoccum mediterrneum Botryospheri dothide Neofusicoccum vitifusiforme Neofusicoccum nonquesitum Tretments Diplodi mutil Diplodi serit Dothiorell ieric Diporthe rhusicol Diporthe neotheicol Control f Figure 1. Averge lesion length (mm) on ttched wlnut rnches fter inocultion with myceli plug of 1 Botryosphericee spp. nd two species of Diporthe. The inocultions were conducted on three wlnut cultivrs in two experiments. Brs topped with different letters indicte tretment mens tht re significntly different (P =.5). Averge lesion dimeter (mm Lsiodiplodi citricol Neofusicoccum prvum Botryospheri dothide Neoscytlidium dimiditum Neofusicoccum mediterrneum c c Neofusicoccum nonquesitum d Diplodi serit Diplodi mutil Diporthe neotheicol Neofusicoccum vitifusiforme Tretments e e e Dothiorell ieric Diporthe rhusicol Figure 2. Averge lesion dimeter (mm) on ttched fruit hulls fter inocultion with myceli plug of 1 Botryosphericee spp. nd two species of Diporthe. The inocultions were conducted on fruit of three wlnut cultivrs in two experiments. Brs topped with different letters indicte tretment mens tht re significntly different (P =.5). f f f Control g Cliforni Wlnut Bord 375 Wlnut Reserch Reports 12

10 Averge lesion length (mm) Chndler Tulre Vin Cultivrs Figure 3. Averge lesion length (mm) on ttched rnches of three wlnut cultivrs fter inocultion with mycelil plug of ech of 1 Botryosphericee species nd two species of Diporthe (verge of two experiments). Brs topped with different letters indicte significnt differences mong cultivrs (P =.5). Averge lesion dimeter (mm) c Chndler Tulre Vin Cultivrs Figure 4. Averge lesion length (mm) on ttched wlnut hulls of three wlnut cultivrs fter inocultion with mycelil plug of ech of 1 Botryosphericee species nd two species of Diporthe (verge of two experiments). Brs topped with different letters indicte significnt differences mong cultivrs (P =.5). Cliforni Wlnut Bord 376 Wlnut Reserch Reports 12

11 Averge lesion length (mm) c c d Aug. 13 Sept. 11 Oct. 1 Oct. 22 Nov. 13 Dte Figure 5. Averge lesion length (mm) on ttched wlnut rnches fter periodic inocultions with mycelil plug of five isoltes of Botryosphericee (Neofusicoccum mediterrneum: 5H53 nd 5C87; N. prvum: 5B6; Botryospheri dothide: 2E55; Lsiodiplodi citricol: 6I35) on three wlnut cultivrs. Brs topped with different letters indicte significntly different mens t P =.5. Averge lesion dimeter (mm) Aug. 13 Sept. 11 Oct. 1 Oct. 22 Dte Figure 6. Averge lesion length (mm) on ttched wlnut fruit hulls fter periodic inocultions with mycelil plug of five isoltes of Botryosphericee (Neofusicoccum mediterrneum: 5H53 nd 5C87; N. prvum: 5B6; Botryospheri dothide: 2E55; Lsiodiplodi citricol: 6I35) on three wlnut cultivrs. Brs topped with different letters indicte significntly different mens t P =.5. Cliforni Wlnut Bord 377 Wlnut Reserch Reports 12

12 Averge lesion length (mm) c c Aug. 13 Sept. 11 Oct. 1 Oct. 22 Nov. 13 Dte Figure 7. Averge lesion length (mm) on ttched wlnut rnches fter periodic inocultions with conidil suspension of ech of five isoltes of Botryosphericee (Neofusicoccum mediterrneum: 5H53 nd 5C87; N. prvum: 5B6; Botryospheri dothide: 2E55; Lsiodiplodi citricol: 6I35) on three wlnut cultivrs. Brs topped with different letters indicte significntly different mens t P =.5. Averge lesion dimeter (mm) Aug. 13 Sept. 11 Oct. 1 Oct. 22 Dte Figure 8. Averge lesion length (mm) on ttched wlnut fruit hulls fter periodic inocultions with conidil suspension of ech of five isoltes of Botryosphericee (Neofusicoccum mediterrneum: 5H53 nd 5C87; N. prvum: 5B6; Botryospheri dothide: 2E55; Lsiodiplodi citricol: 6I35) on three wlnut cultivrs. Brs topped with different letters indicte significntly different mens t P =.5. Cliforni Wlnut Bord 378 Wlnut Reserch Reports 12

13 Volume of wlnut liquid (ml) %) %( %' %% %& $ # 6789:;1< =+;8<1 >?98 "! *+,-.'/ %/ 123-.(& 453-.%/ 453-.(& Dte Figure 9. Volumes of liquid extrct in ml from wlnut fruit hulls ( cm 3 ) in different sesons. Three wlnut cultivrs, chndler, Tulre nd Vin were tested. Extrction of liquid ws conducted y mortl nd pestle on Aug. 25, Sept. 15, Sept. 3, Sept. 15 nd Oct. 3. Wlnut liquid concentrtion ("'!# ("&!# ("%!# ("$!# ("!!#!"'!#!"&!#!"%!#!"$!# 56789:/; <*:7;/ =>87!"!!# )*+",$-",. /1",(-. /1",2! 341",(- 341",2! Dte Figure 1. Concentrtion of liquid extrct from fruit hulls of Chndler, Tulre, nd Vin wlnut cultivrs required in conidil suspension of Neofusicoccum mediterrneum (isolte 5C87) to inhiit 5% of germintion fter 8 hours. Inhiition of conidi germintion tests were conducted t five different dtes. Cliforni Wlnut Bord 379 Wlnut Reserch Reports 12

14 Percentge of wlnut rnches infected #!" '&" '%" '$" '#" '!" &" %" $" #"!" ( )* +,-+./,-+) +12+,/) 34,56* +, 67.) ,56* +, )9467 :,912,/ ;+1<=* )/4* 3,=* )/4 Tretment Figure 11. Incidence of infection of wlnut rnches infested with scle nd without scle fter inocultion with Lsiodiplodi citricol (isol. 6I35), N. prvum (isol. 7E81) nd N. mediterrneum (isol. 5C87). Results were recorded three weeks fter inocultion. Averge lesion length (mm) c d e f g g With scles Lsiodiplodi citricol Neofusicoccum prvum Neofusicoccum mediterrneum Control No scle Tretments Figure 12. Averge lesion length of wlnut rnches infested with scle nd without scle fter inocultion with Lsiodiplodi citricol (isol. 6I35), N. prvum (isol. 7E81) nd N. mediterrneum (isol. 5C87). Results were recorded one week fter inocultion. Brs topped with different letters indicte significnt (P =.5) differences etween lesion length of scle-infested vs. non-infested rnches. Cliforni Wlnut Bord 38 Wlnut Reserch Reports 12