Influence of Temperature, Leaf Wetness Period, Leaf Age, and Spore Concentration on Infection of Pecan Leaves by Conidia of Cladosporiumcaryigenum

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1 cology and pidemiology Influence of Temperature, Leaf Wetne Period, Leaf ge, and Spore Concentration on Infection of Pecan Leave by Conidia of Cladoporiumcaryigenum T.. Gottwald eearch pathologit, USD-S, Southeatern Fruit and Tree Nut eearch Laboratory, P.. Box, Byron, G 31. The author greatly appreciated the technical aitance of aymon. Pate and Jame Stuckey. Mention of a trademark or proprietary product doe not contitute a guarantee or warranty of the product by the USD and doe not imply approval to the excluion of other product that alo may be uitable. ccepted for publication September 194. BSTCT Gottwald, T Influence of temperature, wetne period, age, and pore concentration on infection of pecan leave by conidia of Cladoporium caryigenum. Phytopathology 5: To develop a method for rapidly creening a large number of pecan eedling in a breeding program for reitance to pecan cab, a tudy wa conducted to determine the greenhoue environmental optima for infection by the pathogen, Cladoporium caryigenum. Optimum temperature for infection of pecan foliage ranged 15 to 5 C. Maximum leion development occurred with 4 hr of continuou free foliar moiture. Foliar uceptibility decreaed with increaing age. Maximum foliar uceptibility occurred -1 day after bud break. Foliage became relatively reitant to infection upon reaching full expanion. Leion number and dieae rating increaed directly with inoculum concentration; the highet concentration,.5 X 16 conidia per milliliter, produced the mot leion. However, lower concentration were adequate and more practical to produce for large-cale greenhoue cab creening. dditional key word: breeding for reitance. Scab of pecan [Caryaillinoeni (Wang.) Koch] i a devatating dieae of mot commercial cultivar throughout the outheatern United State. The cab pathogen, Cladoporiumcaryigenum (ll. et Lang.) Gottwald (5) caue evere foliar necroi, twig dieback, leion on the nut huck (involucre), and reduce crop quality (,9). lthough conidia of C. caryigenum are dipered primarily by wind, rain-plah of conidia i a major contributor to localized inoculum diperal (6,,11). Moiture in the form of rain, fog, or dew i required for ucceful infection (3,,11,13,14). The requirement of foliar moiture for infection ha been demontrated previouly. Demaree and Cole (4) were able to initiate infection uccefully by bruhing a conidial upenion of inoculum collected the field onto pecan let and encloing the let in double glaine bag. Convere (1) later demontrated the requirement for free moiture when he prayed or dripped an leion onto or the huck collected upenion inoculum tree after inoculated developed (1). Symptom potted tree were atomized with water for 4 hr at C. He reported infection on kept at 19- C during incubation for 14- day. Latham (1) later wa able to achieve ucceful infection of greenhoue-grown cultivar Schley pecan tree prayed with a conidial upenion containing 3. X 16 pore per milliliter collected cabby pecan leave (1). fter inoculation, tree were held in darkne at 5 C and 1% relative humidity for 1 hr. Pecan foliage i highly uceptible to C. caryigenum when immature but become increaingly reitant a it mature (1). Later, reearcher were able to correlate high foliar trichome denity and low paliade parenchyma phenolic content with tiue uceptibility (15). Trichome denity decreae and phenolic content increae with foliar age. The gricultural eearch Service of the U.S. Department of griculture ha etablihed a pecan breeding program to elect and improve pecan germ plam. The initial creening of pecan progeny in thi program i an elimination baed on cab uceptibility. Becaue an anticipated, to 5, eed will be creened each year, a rapid creening method i needed to handle the large number to be evaluated. However, inufficient data on infection procedure and environmental optima are available on which to bae a creening procedure. The objective of thi tudy wa to determine the optimum environmental condition, age, and inoculum concentration for infection of pecan foliage by C. caryigenum. The data reported herein will be the bai for a rapid greenhoue creening program. MTILS ND MTHODS Inoculum preparation. Conidia of C. caryigenum were harveted 6-wk-old culture on oatmeal agar grown at 4 C with a 1-hr light/dark regime in a walk-in growth chamber. Light wa provided by fluorecent light (Wetinghoue F4/gro-lite, mounted --3cmabovethepetriplate)thatprovidedaphoton flux denity of 1,5 M. m-. ec-1 at the colony urface. Humidity wa held at "-5%. Five milliliter of terile ditilled deionized water were pread onto the urface of each petri plate and the colony urface wa bruhed lightly with a tiff artit' bruh to looen and upend the conidia. The upenion wa centrifuged at 1, rpm for 5 mi, the upernatant wa decanted, and the pore pellet wa reupended in terile, ditilled, deionized water. Centrifugation wa repeated twice to remove culture nutrient, taling product, etc. The final pore concentration wa determined via hemacytometer and adjuted to that needed for pecific experiment. Iolate 1-1 F, a pathovar epecially adapted to cultivar Wichita pecan, wa ued in all experiment becaue it wa highly virulent on greenhoue-grown pecan eedling and porulated more profuely in culture than other iolate. Temperature veru wetne period. upenion of.65 wa atomized with an artit' air bruh X 1' conidia per milliliter The publication cot of thi article were defrayed in part by page charge payment. Thi article mut therefore thereby marked advertiement" in accordance with 1 U.S.C.1 toperated at 1.4 kg/ cm ( pi), onto both adaxial and abaxial urface of 6, -wk-old greenhoue-grown pecan eedling (eed Thi article i in the public domain and not copyrightable. It may be freely reprinted with cutomary crediting of the ource. The merican Phytopathological Society, PHYTOPTHOLOGY ource, cultivar Wichita) to run-off. Inoculated eedling were placed in dew chamber (model F-6DL; Percival Manufacturing Co., Boone, I), 1 per chamber, programmed for an ambient

2 temperature of 1, 15,, 5, 3, or 35 C, 1% H, free dew formation, and total darkne. Tree, elected at random for poition in the chamber, were removed the chamber after,,4, 6,9, 1, 1, 4, 36, or 4 hr in replicate of 1. Following thi wetne period all tree were returned to the greenhoue and placed on a and bed where they were held in air-conditioned temperature of 4- C in a polyethylene high-humidity encloure (5-9% H) to promote leion development. fter a 1-day incubation period, tree were removed the encloure and the econd, third, fourth, and fifth (econd oldet, fifth younget) leave were rated on a 1-5 cale for percent dieae (1 = no leion; = few mall brown fleck <1. mm; 3 = numerou leion, coniting of brown fleck mixed with mall black leion, 1- mm; 4 = mall well-developed black leion, 1- mm; and 5 = large well-developed preading black leion, >. mm). Thee leave were then removed the eedling, the leion on each were counted, and the area wa meaured with a -area meter (model Li-3 1; Li-Cor, Inc., Lincoln, N) and number of leion per quare centimeter were calculated. The firt or -mot wa much maller than other leave and often ditorted; therefore, it wa not included. xperimental deign wa a 6 X 1 factorial (ix temperature and 1 wetne period) replicated 1 time. ffect of age and infection. Numerou pecan eed were ed in.5-cm-diameter peat mo cup and germinated in an air-conditioned greenhoue, 4- C. Seedling were grouped by date of emergence into group of each and only the mot uniform were utilized in the tudy. ach group of wa then inoculated with conidia (concentration.5 X l' pore per milliliter collected and treated a decribed above) at 1,, 14, 1, or day potemergence. ge of individual leave (day budbreak) on each at time of inoculation wa determined by referencing each to it individual date of bud break. Plant were randomized in Percival dew chamber following inoculation for4 hrat5 C, 1% H, free dew formation, and totaldarkne. Following the wetne period, all were returned to the greenhoue and held for 3 wk in the high-humidity encloure, rated for dieae, and the area were meaured a decribed above, ffect of pore concentration on infection. To determine the optimum inoculum pore concentration for infection, group of 3-wk-old greenhoue-grown pecan eedling (eed ource, cultivar Wichita) were inoculated with, 3, 16,, 4. X I',.X 14, 1. X 15, 5. X 1', or.5 X 16 pore per milliliter of terile ditilled water reulting a dilution erie prepared by uing the inoculum preparation technique decribed above. ll were atomized with inoculum via the airbruh technique dicued previouly. Immediately following inoculation, all were randomized in Percival dew chamber at 5 C, 1% H, for 4 hr. Plant were then returned to the greenhoue andbed, incubated, rated for dieae, and the leave were meaured a previouly decribed. Statitic. ll three tudie were replicated at leat once. Data were ubjected to analyi of variance and difference among treatment mean were detected by uing Duncan' multiple range tet. Data on the effect of pore concentration were alo ubjected to linear regreion. Graphic analyi wa performed with SSGraphic (Graphic package of Statitical nalyi Sytem, SS Intitute, Cary, NC). SULTS Leion development. In all tudie, pecan cab leion firt became noticeable -9 day after inoculation. Leion continued to develop for 14-1 day at which time they had attained full ize and topped expanding. fter 1 day, leion ranged minute brown fleck with no apparent diruption of the epidermi and no external evidence of hyphae (dieae rating = ) to luxuriant erumpent outgrowth of hyphae and conidiophore laden with numerou catenulate chain of blatoconidia (dieae rating = 5). ffect of temperature and wetne period on infection. Dieae developed at all temperature--wetne-period combination teted. Leion number generally increaed with increaing wetne period. ',w leion developed after only hr of continuou dew; however, maximum leion per quare centimeter urface area (.365) occurred after 4 hr of wetne (Fig. 1). Total number of leion produced at 1, 15, 3, and 35 C were tatitically the ame (P =.5) for all wetne period. Maximum leion production occurred after 3-4 hr of wetne at C and 4 hr of wetne at 5 C. Data rated for dieae intenity and data of actual leion count produced very imilar reult; however, temperature appeared le critical for maximum dieae production. LSIONS P SO CM HS LF WTNSS 5 TMPTU C) Fig. 1. The effect of temperature and wetne period on the total number of pecan cab leion per quare centimeter of urface area of -wk-old greenhoue-grown pecan eedling (eed ource, cultivar Wichita). Note ome infection took place after hr of wetne, but maximum leion number reulted C and 4 hr of continuou wetne. Vol. 5, No.,

3 Temperature of 15,, and 5 C during 4 hr of continuou wetne all reulted in imilar maximum dieae rating of (Fig. ). Few leion (Fig. 1) and little dieae (Fig. ) developed during wetne period of <9 hr. Increaing the wetne period up to or beyond 9 hr tended to timulate an overall increae in leion number and dieae rating at all temperature teted (Fig. 1 and, note teplike increae at 9 hr of wetne); however, there wa no tatitical difference (P =.5) between 9 and 36 hr of wetne at any temperature teted. definite dieae increae wa demontrated when wetne period were extended beyond 36 hr within the optimum temperature range of 15-5 C. ffect of age on infection. The firt inoculation to tet the uceptibility of leave of variou age to optimum condition of temperature and wetne (5 C and 4 hr of wetne) wa conducted when the firt (terminal) eedling bud had been broken for - I day. The outermot at thi time wa often repreed in ize and oddly haped and wa therefore not ued. The econd and third outermot leave were moderately uceptible to inoculum of C. caryigenum at thi time while the fourth and fifth leave were generally unexpoed and cloely appreed to the hort apex. They were more protected and maller in urface area (Table 1) and therefore appeared lightly le uceptible. Inoculation day after bud break when econd and third leave were about one-half expanded demontrated increaed uceptibility to inoculum of C. caryigenum. Fourteen to 1 day after bud break, the firt leave to unfold (econd and third leave) were nearly fully expanded and omewhat le uceptible, however, thoe leave jut lightly later to unfold (fourth and fifth leave) were in their mot uceptible condition. The outermot leave (econd and third) of the terminal bud became completely reitant to infection (after 14 and day, repectively) while the inner leave were till highly uceptible (Table 1). When the uceptibility of entire wa conidered, there wa no tatitical difference among group of inoculated at variou day after bud break due to the variability of individual uceptibility over time and ultimate urface area (Table 1). However, there wa a trend of increaed dieae uceptibility between and 1 day after bud break which i a reflection of thi ame trend for individual poition. ffect of inoculum concentration on infection. roughly fivefold dilution erie.5 X 16 to 3 conidia per milliliter of terile, ditilled water wa prayed on pecan eedling at the optimum temperature-wetne period of 5 C-4 hr decribed above. The number of leion per quare centimeter urface area increaed with inoculum concentration for each poition and for the a a whole (Table ). The relationhip between the natural logarithm of inoculum concentration and number of leion per quare centimeter of urface area wa fairly linear ( =.) for concentration of to 5 X l 5 conidia per milliliter. Leion number increaed more than fivefold when the inoculum concentration increaed 5 X l' to.5 X 16 conidia per milliliter. The data were therefore ubjected to two regreion analye. The firt demontrated a ignificant repone of number of leion per to increaing inoculum concentration to 5. X 15 conidia per milliliter. The econd analyi demontrated the increae in repone (teeper lope) between 5. X 15 and.5 X 16 conidia per milliliter (Fig. 3). The average dieae rating wa linearly related to the natural log of pore concentration over the entire range of pore concentration teted (r =.9) (Fig. 4). DISCUSSION The requirement of wetne period caued by dew, fog, and miting rain for infection of pecan by C. caryigenum ha been uggeted many time (1,1-1). My reult indicate that infection increae with duration of wetne. Convere decribed the optimum temperature for conidial germination a -5 C (). My experiment with eedling pecan have demontrated that optimum temperature for infection wa C when combined with a 4-hr wetne period. Succeful infection were obtained within the temperature range of 15-5 C when leave were wet for 4 hr. lthough continuou 4-hr wetne period are unuual in the orchard, -3 day of intermittent hower are not uncommon and can create wetne period approaching 4 hr. xtended potinoculation wetne period apparently did not wah pore off the foliage to any appreciable extent a the longet wet period promoted the mot leion. n increae in leion number with increaing wetne period could be a repone to delayed pore germination. Not all pore of C. caryigenum wahed petri dih culture may be in a germinable tage when dilodged their conidiophore. Thee latent pore may FOLI DISS TING 3.6 / = 3 HS TMPTU CSC) LF WTNSS Fig.. The effect of temperature and wetne period on pecan cab dieae a etimated by foliar dieae rating. (ating cale: 1 = no leion; = few mall brown fleck < 1. mm; 3 = more numerou leion-brown fleck mixed with mall black leion; 4 = mall well-developed black leion 1- mm; and 5 = large well-developed preading black leion >. mm.) Tet wa conducted on -wk-old greenhoue-grown pecan eedling (eed ource, cultivar Wichita). 19 PHYTOPTHOLOGY

4 germinate only after reiding for a period of time on the urface in the preence of free moiture. For the purpoe of creening large number of greenhoue eedling, the temperature range for maximum infection of 15-5 C i mot ueful and not difficult to maintain in an air-conditioned greenhoue bay. The preence of free foliar moiture can be eaily provided by commercial greenhoue fogging equipment. The relative reitance of older pecan foliage to infection by C. caryigenumha been previouly noted (1). My reult indicate that uceptibility of pecan foliage decreaed rapidly with age. Foliage wa mot uceptible ---1 day after bud break. Prior to day after bud break, the foliage ha very limited urface area, and therefore, even though highly uceptible, only a few infection can occur and thee become pread out a the expand. fter 1 day, the leave are approaching full ize, cuticle formation catche up with expanion, and there i a depoition of phenolic ubtance repreent a mixture of everal train or genotype. In a creening program, it i deirable to creen againt preteted, highly virulent, conitent inoculum a pathovar (or pathovar) pathogenic to the eed parent cultivar (or cultivar). For uch creening, inoculum.5 L N P 3 in the paliade parenchyma layer of the blade (15). ny or all C of thee change may make the reitant to infection. In addition, the chemical compoition of urface exudate change with age (T.. Gottwald and B. W. Wood, unpublihed). Thi change in water-oluble chemical contitution may well affect the germination and/or viability of pore of C. caryigenum on the phylloplane. When uceptibility of the entire eedling i conidered rather than that of the individual, optimum infection can be achieved when inoculation are made '-' day after bud break. Thi i a mot fortunate circumtance becaue large number of eedling can be rapidly creened for cab uceptibility with a very hort turnaround time. lthough field inoculum ha been ued uccefully in the pat in greenhoue tudie (1), it i not alway readily available. In addition, ince uch inoculum i collected numerou leion, it viability and pathogenicity i often quite variable and may Y-.95X to II LN SPO CONCNTTION IS Fig. 3. The effect of inoculum concentration (logarithmically tranformed) on the number of pecan cab leion per quare centimeter of urface area. Note increae between lat two point on curve which correpond to 5. X 16 and.5 X 16 conidia per milliliter. Tree were ubjected to 5 C and a wetne period of 4-hr potinoculation. Note that the regreion coefficient i ignificantly greater than, indicating that there i a ignificant repone in the number of cab leion per quare centimeter of urface area to pore concentration..5 v.5- G. TBL 1. The effect of pecan age on infection by Cladoporium caryigenumx Leion per quare centimeter Day after bud break nd y.-a (4) rd 4th 5th.4a (V).119c ().39c () D I I.e- verage per T. 93 Y N G.6aa._-.419a(½/).366a(V().53abc(¼).39bc(%).44a. b (34).66 a (1/).9 ab (½) 1.9 a (¼).34 a. b (F).5 a (3/4) a (1/) 1.39 ab (V).311 a S I LN SPO CONCNTTION. b (F). a (F).455 bc (34).943 bc (½).14 a._b_(f)._a_(f)_. 55_b (_)_.943 be_(_.14 a XGreenhoue grown pecan eedling (eed ource, cultivar Wichita) were Fig. 4. The effect of inoculum concentration (logarithmically tranformed) on pecan cab dieae rating of eedling pecan cultivar Wichita. Potinoculation temperature wa 5 C during a 4-hr wetne period. inoculated 1,, 14, 1, or day after firt bud break. Dieae determination were made for each 3 wk after inoculation. Mean followed by the ame letter are not ignificantly different at P =.1. 'Term in parenthei following leion count denote general overall tage, ie, = newly emergent ; V = one-quarter fully expanded, ½ = half fully expanded, 3/4 = three-quarter fully expanded, and F = fully expanded. (ating cale: 1 = no leion; = few mall brown fleck, <1. mm; 3 = more numerou leion-brown fleck mixed with mall black leion; 4 = mall well-developed black leion, 1- mm; and 5 = large welldeveloped preading black leion, >. mm.) Note that the regreion coefficient i ignificantly greater than, indicating that there i a ignificant repone in number of leion per quare centimeter urface to dieae rating. TBL. The effect of inoculum concentration on infection of pecan foliage by Cladoporium caryigenum' Inoculum concentration (pore per ml) 4th Leion per quare centimeter 5th 6th verage per Leaf rating 4th 5th 6th verage per X 1' X 1' X X 1' X 'Greenhoue-grown pecan eedling (eed ource, cultivar Wichita) were inoculated with nine concentration of conidia of C. caryigenum when - 14 day old. Dieae determination were made -3 wk after inoculation. Vol. 5, No.,

5 produced in the laboratory may therefore be uperior to inoculum 4. Demaree, J. B., and Cole, J Behavior of Cladoporium collected directly the field. effuum (Wint.) Demaree on ome varietie of pecan. J. gric. e. lthough thi tudy indicated that the highet inoculum 3: concentration teted reulted in the highet leion count, 5. Gottwald, T Taxonomy of the pecan cab fungu production of large quantitie of inoculum at thi concentration Cladoporium caryigenum. Mycologia 4:3-39. (.5 X 16 conidia per milliliter) i extremely time conuming and 6. Gottwald, T Spore dicharge by the pecan cab pathogen, Cladoporium caryigenum. Phytopathology : difficult to achieve with preent technique. More eaily obtained. Gottwald, T.., and Bertrand, P. F. 19. Pattern of diurnal and concentration of X l5 conidia per milliliter are quite eaonal airborne pore concentration of Fuicladium effuum and it adequate to produce ufficient inoculum preure for practical impact on a pecan cab epidemic. Phytopathology : large cale creening of pecan eedling.. Gottwald, T.., and Bertrand, P. F ffect of time of inoculation eult of thi tudy can be ued to develop tandard technique with Cladoporium caryigenum on pecan cab development and nut to ae the uceptibility of progeny the USD/ S pecan quality. Phytopathology 3:14-1. breeding program to pecan cab. Such a creening program would 9. Hunter, Influence of cab on late eaon nut drop of pecan. Plant Di. 6:6-. allow election of reitant and erve to eliminate large 1. Latham,. J Some factor influencing pecan cab Fuicladium number of prior to ing in the field for further effuum development. uburn gric. xp. Stn. Highlight gric. e. evaluation. However, further tudie including the effect of mixed 6:. inoculum everal different pathovar and the development of 11. Latham,. J. 19. ffect of ome weather factor and Fuicladium entire greenhoue bay climate and wetne period control will effuum conidium diperal on pecan cab occurrence. Phytopathology be needed before uch a creening program can be implemented. : Latham,. J Control of Cladoporium caryigenum on pecan LITTU CITD leave and nut huck with propiconizal (CG-645). Plant Di. 6: Convere,. H Phyiological pecialization of Fuicladium 13. Nolen, Pecan cab. Page 11-1 in: Fla. gric. xp. Stn. effuum and it evaluation in vitro. Phytopathology 5: Bull Convere,. H The production and germination of conidia of 14. Valli, V. J Weather condition a related to pecan cab infection. Cladoporium effuum (Wint.) Demaree in the laboratory. (btr.) Proc. S.. Pecan Grower oc. 5:5-. Phytopathology 46: Wetztein, H. Y., and Spark, D natomical tudie of cultivar 3. Demaree, J. B Pecan cab with pecial reference to ource of the and age-related cab reitance and uceptibility in pecan leave. J. early pring infection. J. gric. e. 3: m. Soc. Hortic. Sci. 1: PHYTOPTHOLOGY