DISEASE AND PEST MANAGEMENT

HORTSCIENCE 50(8):1183–1186. 2015. transmission from infected scions through the application of a hot-water treatment to dormant scions before grafting (Sanderlin Susceptibility of Some Common Pecan and Melanson, 2008). The application of the hot-water treatment to pecan rootstock has Rootstocks to Infection by Xylella not been tested and may be challenging because of the size of the rootstocks, and fastidiosa the need to treat the rootstock or grafted tree while dormant before transplanting into an Randy S. Sanderlin1 orchard. Louisiana State University Agricultural Center Pecan Research-Extension Pecan rootstocks are produced from Station, 10300 Harts Island Road, Shreveport, LA 71115 open-pollinated seed of grafted cultivars. Certain cultivars are preferred as sources of Additional index words. Carya illinoinensis, pecan bacterial leaf scorch, pathogen graft seed for rootstocks based on size of the nuts, transmission, mechanical inoculation vigor of growth, and the effects on early tree growth and nut production following graft- Abstract Carya illinoinensis . Pecan [ (Wangenh.) K. Koch], a hardwood tree native to ing to scions (Grauke and Thompson, 1995). North America, is grown for commercial production of nuts in southeastern, central, and The use of pecan rootstocks resistant to western regions of the United States. Pecan is also grown commercially in Mexico, South infection by X. fastidiosa combined with Africa, Australia, and some countries in South America. Pecan trees can be infected by hot-water treatment of scions may provide Xylella fastidiosa the broad host range xylem-limited bacterial pathogen Wells et al. a way for commercial pecan nurseries and Infection incites a leaf scorch disease that can cause significant defoliation, reduced tree pecan growers to establish new trees free of growth, and lower nut yield. Pecan cultivars are clonally propagated onto rootstocks the pathogen. Rootstock resistance to X. X. fastidiosa grown from open-pollinated seed of selected cultivars. is transmitted at fastidiosa infection has been identified in a high frequency from infected rootstocks into newly developing grafted trees. some other agricultural commodity hosts of Rootstocks resistant to infection would be beneficial to pecan nurseries and pecan the pathogen including, grapevine, almond, producers to prevent infection of young trees through grafting, especially when and peach (Gould et al., 1991; Gubler et al., combined with hot-water treatment of scions to eliminate the pathogen. Some common 2008; Ledbetter and Rogers, 2009). Pres- rootstocks were tested for variation in susceptibility to infection using mechanical ently, there is no information available on the inoculation with the pathogen. No outstanding level of resistance to infection was susceptibility of rootstocks used by the pecan detected among the seven rootstocks tested. The rootstocks from ‘Curtis’, ‘Elliott’, and nursery industry to infection by X. fastidiosa. ‘Riverside’ were less susceptible than one standard rootstock in the test (‘VC1-68’) and The objective of this research was to test less susceptible than highly susceptible ‘Cape Fear’ rootstock. Conversely, the rootstocks some of the pecan rootstocks commonly from ‘Apache’, ‘Moore’, ‘Stuart’, and ‘VC1-68’ seed had a level of susceptibility to used by commercial nurseries for their de- infection comparable to ‘Cape Fear’ and perhaps are not the best choice for rootstocks in gree of susceptibility to infection by the X. fastidiosa geographic areas where is prevalent. The results of this research suggest that PBLS pathogen. This report provides the X. fastidiosa there is variation in rootstock susceptibility to infection by . The use of first data on pecan rootstock susceptibility mechanical inoculation may facilitate identification of susceptibility categories of pecan to X. fastidiosa. rootstocks to the pecan bacterial leaf scorch pathogen. Materials and Methods Pecan [C. illinoinensis (Wangenh.) K. In pecan, the disease causes defoliation, Seven rootstock types grown from open- Koch], a tree species native to the Mississippi yield loss, and reduced growth of trees pollinated seed of the following cultivars River floodplain of North America, is culti- (Sanderlin and Heyderich-Alger, 2003). As were used in the study: ‘Apache’, ‘Curtis’, vated for nut production across the south- with other hosts of this bacterium, the path- ‘Elliott’, ‘Moore’, ‘Riverside’, ‘Stuart’, and eastern United States, and other states ogen is transmitted to pecan through feeding ‘VC1-68’. Trees grown from seed of ‘Cape including Texas, Oklahoma, Kanas, New by certain members of the Cicadellidae (leaf- Fear’, which is not typically used as a root- Mexico, Arizona, and California (Graham hoppers) and Cercopidae (spittlebugs) insect stock, were included as a standard for com- and Gibson, 2005). Commercial pecan pro- families (Redak et al., 2004; Sanderlin and parison because of its known high degree of duction also occurs in Mexico, South Africa, Melanson, 2010). In addition to insect trans- susceptibility as a cultivar and as nongrafted several South American countries, and Aus- mission, the pathogen can be transmitted in trees grown from ‘Cape Fear’ nuts (Littrell tralia (Geisler, 2011). Pecan is susceptible to pecan through infected rootstocks or scions and Worley, 1975; Sanderlin and Heyderich- infection by X. fastidiosa ssp. multiplex (Sanderlin and Melanson, 2006). Once in- Alger, 2000). Seeds for production of root- resulting in the development of pecan bacte- fection is established, the bacterium tends to stocks from cultivars of ‘Apache’, ‘Elliott’, rial leaf scorch (PBLS) disease (Melanson become systemic in trees with chronic dis- ‘Moore’, ‘Riverside’, and ‘VC1-68’ were et al., 2012). The multiplex subspecies has ease development. Because there is no effec- supplied by the U.S. Department of Agricul- a large host range, including numerous hard- tive treatment to eliminate the pathogen from ture Pecan Breeding and Genetics Station at wood species, and causes disease in a variety infected trees, pecan trees infected through Somerville, TX. Seeds of cultivars ‘Cape of agriculturally important species including grafting will be affected by the disease Fear’, ‘Curtis’, and ‘Stuart’ were collected fruit and nut trees in the United States, and throughout their life span, which can be from trees at the LSU AgCenter Pecan was recently reported to have infected olive several decades. In new orchards, graft trans- Research-Extension Station, Shreveport, trees in Italy (European Food Safety Author- mission can provide the primary inoculum of LA. The seeds were stored near 4 C through ity, 2013; Hopkins and Purcell, 2002). X. fastidiosa, which may be subsequently the winter in moist vermiculite for stratifica- spread by insect vectors. Without the estab- tion, and a single seed was planted into 25 · lishment of the pathogen in an orchard 25 cm (9.5-L volume) plastic pots contain- through graft transmission, an orchard may ing MetroMix 902 (Sungro, Agawam, MA) Received for publication 4 Mar. 2015. Accepted for publication 29 June 2015. escape several years before the pathogen is plant growth medium in Mar. 2013. The trees I thank Dr. L.J. Grauke for providing seed for introduced by insect vectoring (Sanderlin, were grown for 1 year in a greenhouse and rootstock production. unpublished data). inoculated in 2014. At the time of inocula- 1Corresponding author. E-mail: rsanderlin@agcenter. It should be possible to reduce the tion, most trees were between 30 and 40 cm lsu.edu. incidence of PBLS by reduction of graft in height.

HORTSCIENCE VOL. 50(8) AUGUST 2015 1183 Trees were observed for PBLS symptoms PBLS symptoms from their inoculation date types including ‘Cape Fear’. The rootstocks and assayed for the pathogen by enzyme- until the trees went dormant in the green- from ‘Apache’, ‘Moore’, and ‘Stuart’ were linked immunosorbent assay (ELISA) before houses in late December. also not significantly different from ‘Cape inoculation to confirm that X. fastidiosa was Tissue for ELISA of the inoculated trees Fear’. Even though rootstocks from seed of not detectable in the plants by serological was collected initially at 9- to 12-weeks post ‘Apache’ and ‘Moore’ had a lower inci- assay. For ELISA, the two or three oldest inoculation. Additional assays were done as dence than ‘Cape Fear’ type rootstocks, leaves were removed from each tree and the symptoms developed. Trees in each of the they still each had higher than a 50% in- rachises used for assay. All ELISAs conduct- three inoculation periods that had not pre- fection level. The rootstock grown from ed throughout the study were performed with viously assayed positive for infection were ‘Curtis’, ‘Elliott’, and ‘Riverside’ had a sig- a commercial kit for X. fastidiosa (Agdia given a final ELISA at 17 to 22 weeks nificantly lower incidence of infection than Inc., Elkhart, IN). The preinoculation assays postinoculation. Five noninoculated control ‘Cape Fear’ and ‘VC1-68’; but were not were done within a week before inoculation. trees of each rootstock type were assayed 18 lower than any of the other rootstocks tested For each inoculation date, the inoculum weeks after the first X. fastidiosa inoculation (Table 1). was started from cultures stored at –80 Cin date. The majority (86%) of the trees of that 30% glycerol. Cultures were regenerated on For statistical analysis, inoculation dates were positive for infection by ELISA either a single petri plate of periwinkle wilt agar were treated as experimental blocks, and the had symptoms of PBLS when the positive (PWA), and then transferred to fresh media experimental design was a randomized com- assay tissue was collected or developed several times to increase bacterial quantity. plete block. Data were analyzed with the symptoms later after the positive assay. There Each inoculum preparation was made up of Tables function of SAS version 9.4. The were 10 trees that assayed positive for X. the growth of three isolates produced in Cochran–Mantel–Haenszel test was used for fastidiosa infection by ELISA that were not separate cultures. The isolates had been mean comparison to determine significant recorded with PBLS symptoms during the originally cultured from ‘Cape Fear’ pecan differences between infection values of the duration of this work. Within rootstock type in 2005 and verified to be X. fastidiosa ssp. rootstocks. the number of infected trees that failed to multiplex (Melanson et al., 2012). Cultures show symptoms during the observation pe- were maintained in darkness near 28 C. Results riod was 0 to 3. Inoculum was prepared by suspending the growth from 10- to 12-d-old cultures (usually All of the 120 trees inoculated with X. Discussion five plates of each isolate) in 2.5 mL of sterile fastidiosa tested negative for infection by molecular grade water. Inoculum concentra- ELISA before inoculation, and no PBLS The work reported here serves as a starting tion was determined by serial 10-fold dilution symptoms were observed on any trees before point in the attempt to develop knowledge on plating on PWA. inoculation. Throughout the test, no symp- the susceptibility of pecan rootstocks to in- The inoculation procedure consisted of toms of scorch were observed on any of the fection by X. fastidiosa. Ideally, if rootstocks placing five 10-mL quantity drops of inocu- control trees in the greenhouse, and the five with useful levels of resistance to infection lum along the main stem of the tree in or near control trees of each rootstock tested by can be identified, their use will reduce the the current season’s growth. A dissecting ELISA for the pathogen were negative for probability of nursery produced trees being needle was pushed through the drop into infection. infected at the time of sale as a result of graft the stem. The needle was left in place until The inoculum concentration for the 21 transmission through infected rootstocks. the drop was absorbed into the stem, usually July and 5 Aug. inoculations was 108 The results indicate there are differences less than a minute. Because the inoculations colony-forming units (cfu)/mL, and for the across open-pollinated rootstock types in were made in the afternoons of hot summer 29 Aug. inoculation date the concentration susceptibility to infection by X. fastidiosa. days, an attempt was made to maintain the was 107 cfu/mL. The 10–1 dilution, which Under natural conditions, trees to be inoculum at a moderate temperature by was maintained at room temperature during used for rootstocks would be infected by keeping the inoculum vial in a Styrofoam inoculation and applied to PWA after the tree insect transmission of the pathogen. In this container with a layer of ice that was covered inoculation was finished, always produced test, infection challenge was by mechanical by 1.5 cm layer of cotton cloth. The 10–1 a concentrated film of bacterial growth, in- inoculation with high bacterial concentra- dilution of the inoculum, which was kept at dicating that the viability of the pathogen tions. It is not known exactly how suscepti- room temperature during the inoculation suspension was maintained through the 2.5- bility to mechanical inoculation compares procedure, was plated on PWA following to 3-h inoculation time. with susceptibility to vector transmission. the inoculation period to verify that inoculum Symptoms on inoculated trees generally Plant species that may not be naturally viability could be maintained for the length began to develop within 2 months; however, susceptible to infection by a specific sub- of the procedure. as is typical of PBLS, the time of symptom species of X. fastidiosa may occasionally be Trees were removed from the greenhouse development on individual trees was vari- infected with that subspecies through me- and placed outside in full sunlight before able and occurred over a period of 10 (four chanical inoculation (Sanderlin, unpub- inoculation to increase transpiration. One tree trees) to 15 weeks (one tree). All eight of lished data). Mechanical inoculation may of each rootstock type was inoculated fol- the pecan rootstocks were susceptible to therefore present a severe challenge of lowed by a second of each type and this order infection by X. fastidiosa as determined by a plant’s inherent resistance system. Conse- continued until five trees of each rootstock symptoms and ELISA. But there were sta- quently, resistance detected through me- were inoculated. Each inoculation procedure tistical differences in the numbers of trees chanical inoculation may actually be at took 2.5 to 3 h. Trees stayed outside for about infected by the rootstock type (Table 1). a higher functional level in nature. Insect an hour after inoculation of the final tree and The rootstock grown from seed of ‘VC1-68’ transmission efficiency studies with X. fas- then all trees were returned to the green- commonly used by nurseries to produce tidiosa have demonstrated a wide range of house. Three sets of inoculations were per- trees for use in the western U.S. pecan transmission rates and are affected by nu- formed with inoculations on 21 July, 5 Aug., production region with 14 of 15 inoculated merous variables (Redak et al., 2004), but and 29 Aug. 2014. At least 15 noninoculated plants determined to be infected was in- generally insect transmission occurs at trees of each rootstock type, maintained in fected at a statistically higher percentage a lower rate than infection by mechanical the greenhouse, served as control trees, ex- than five of the standard rootstocks and was inoculation (Almeida and Purcell, 2003; cept for the ‘Riverside’ rootstock that had comparable in number of infected trees to Rashed et al., 2011). In this test, 73% of only five control trees because of poor seed the highly susceptible ‘Cape Fear’ seed trees grown from open-pollinated ‘Cape germination. rootstock (Table 1). Symptom production Fear’ seed were infected by mechanical Inoculated trees and control trees were on ‘VC1-68’ rootstock was often more rapid inoculation compared with an insect trans- observed at least weekly for development of and often more severe than on the other mission rate of 11% to 22% when various

1184 HORTSCIENCE VOL. 50(8) AUGUST 2015 Table 1. Infection incidence of pecan rootstock types mechanically inoculated with Xylella fastidiosa ssp. disease than trees grafted to the more multiplex.z susceptible rootstock type of ‘Apache’ Rootstocky Inoculation 21 Julyx Inoculation 5 Aug. Inoculation 29 Aug. Total (Sanderlin, unpublished data). The lack of Apache 4/5w 4/5 1/5 9/15 abcv resistance transfer from rootstocks to scions Cape Fear 3/5 5/5 3/5 11/15 ab also exists in grapevine (Gubler et al., 2008; Curtis 1/5 2/5 1/5 4/15 c Xu et al., 2002). Nonetheless, the produc- Elliott 1/5 4/5 1/5 6/15 c tion of trees initially free of infection from Moore 2/5 3/5 3/5 8/15b c graft transmission through infected root- Riverside 2/5 3/5 0/5 5/15 c stocks and pathogen free scions should be Stuart 2/5 3/5 2/5 7/15b c VC1-68 5/5 5/5 4/5 14/15 a an advantage in new orchard establishment and may result in several years of growth zInfection was determined by a positive assay for X. fastidiosa with a commercial ELISA kit. yRootstocks grown from open-pollinated seed of indicated cultivars. and nut production before the pathogen is xThe date that trees were inoculated in 2014. introduced into the orchard by insect vector wThe numerator is the number of infected trees and the denominator is the number of trees inoculated with transmission. a suspension of the bacterium. vMeans followed by different letters are significantly different by the Cochran–Mantel–Haenszel test (P # Literature Cited 0.05). Almeida, R.P. and A.H. Purcell. 2003. Biological traits of Xylella fastidiosa strains from grapes vectors were exposed to infected pecan trees incidence of infection of the same rootstocks and almonds. Appl. Environ. Microbiol. then transferred to trees from open-pollinated used in the test reported here. Cultivar 69:7447–7452. European Food Safety Authority. 2013. State- ‘Cape Fear’ seed (Sanderlin and Melanson, Apache seed rootstock trees had the highest ment of EFSA on host plants, entry and 2010). incidence of PBLS with 42% of its trees spread pathways and risk reduction options Mechanical inoculation has been used infected, followed by trees from seed of for Xylella fastidiosa Wells et al. EFSA previously to evaluate pecan cultivar and ‘Riverside’ (33%), and ‘Moore’ (19%). Journal. 11:3468. seedling susceptibility with results similar The nongrafted trees of ‘Curtis’ and ‘Elliott’ Garcia, A.L., S.C. Torres, M. Heredia, and S.A. to an orchard survey for the disease (Sanderlin, seed had no trees identified with PBLS after Lopes. 2012. Citrus responses to Xylella 2005). Mechanical inoculation has also 10 years in this orchard. These data correlate fastidiosa infection. Plant Dis. 96:1245– been used to identify resistant rootstock in well with the results of this inoculation test 1249. citrus. A citrus rootstock resistant to citrus in which the rootstocks from ‘Curtis’ had the Geisler, M. 2011. Pecans. Agricultural Marketing Resource Center. 4 Feb. 2015. . tified through mechanical inoculation tests. inoculation. The generally greater infection Gould, A.B., W.J. French, J.H. Aldrich, B.V. The rootstock ‘Rangpur’ did not produce rates with mechanical inoculation compared Brodbeck, R.F. Mizell, III, and P.C. Andersen. symptoms of CVC after it was mechanically with infection by natural insect vectoring 1991. Rootstock influence on occurrence of inoculated and apparently supported only over a decade adds support to the supposi- Homalodisca coagulata, peach xylem fluid a low population of X. fastidiosa. ‘Rangpur’ tion that mechanical inoculation presents amino acids, and concentrations of Xylella was not considered a significant source of a severe challenge to evaluate resistance to fastidiosa. Plant Dis. 75:767–770. inoculum for insect transmission (Garcia infection by X. fastidiosa. Graham, C.J. and A. Gibson. 2005. Louisiana et al., 2012). The data suggest practical benefits would pecan industry: Past and future. Proc. South- eastern Pecan Growers Assoc. 98:72–77. Symptom development is not always result from intensive screening of pecan Grauke, L.J. and T.E. Thompson. 1995. Root- a quick way to detect infection as symptoms rootstocks for resistance to X. fastidiosa. stock development, p. 167–175. In: M.W. can be transitory with symptomatic leaflets With wider screening of additional root- Smith,W.Reid,andB.W.Wood(eds.). falling off and the reoccurrence of foliar stock types, it may be possible to identify Sustaining Pecan Productivity into the 21st symptoms in the same growth period rootstocks with greater resistance to infec- Century. Second National Pecan Workshop delayed or absent. Possible explanations tion than that detected in this initial work. In Proceedings. U.S. Department of Agriculture, for the lack of detection of symptoms in addition, such testing could aid the pecan Agricultural Research Service, Ser. Pub. 14% of the trees that ELISA indicated were industry in identifying rootstocks with ex- ARS-1995-3. infected include failure to observe symp- treme susceptibility to infection. In this Gubler, W.D., R.J. Smith, L.G. Varela, S. Vasquez, J.J. Stapleton, and A.H. Purcell. 2008. UC toms before defoliation of the affected research, rootstocks of ‘VC1-68’ seed were management guidelines for Pierce’s disease of leaflets and potential of ELISA to detect infected at even a higher percentage than grape. 4 Feb. 2015. . symptom development. In previous green- a high level of susceptibility (Sanderlin Hopkins, D.L. and A.H. Purcell. 2002. Xylella house tests, pecan trees recorded as infected and Heyderich-Alger, 2000). Even though fastidiosa: Cause of Pierce’s disease of grape- by ELISA and maintained for a least two ‘VC1-68’ has greater vigor than the other vine and other emergent diseases. Plant Dis. summers always produced symptoms at rootstocks tested with a mean aboveground 86:1056–1066. some point during the two growth periods. growth of 60.8 cm compared with 53.2 cm Ledbetter, C.A. and E.E. Rogers. 2009. Differential It is likely that the symptomless infected for the other seven rootstocks combined susceptibility of Prunus germplasm (Subgenus Amygdalus) to a California isolate of Xylella trees recorded in this work would also pro- when growth was measured during dor- fastidiosa. HortScience 44:1928–1931. duce PBLS symptoms if maintained for mancy in Jan. 2015, it should not be used Littrell, R.H. and R.E. Worley. 1975. Relative another year. as a cultivar for rootstock production in the susceptibility of pecan cultivars to fungal leaf The results from this greenhouse test southeastern United States because of the scorch and relationship to mineral composition using mechanical inoculation are in general higher probability of infection of these of foliage. Phytopathology 65:717–718. agreement with occurrence of PBLS in non- rootstocks in nurseries. Melanson, R.A., R.S. Sanderlin, A.R. McTaggart, grafted rootstock trees in an orchard that was Resistance to X. fastidiosa infection in and J.H. Ham. 2012. A systematic study reveals monitored for spread of PBLS for 10 years pecan rootstocks does not appear to be that Xylella fastidiosa strains from pecan are (Sanderlin, unpublished data). The orchard conferred to scions grafted onto to them. In part of X. fastidiosa subsp. multiplex. Plant Dis. 96:1123–1134. contained a mixture of cultivars and non- the orchard study of PBLS spread where Rashed, A., M.P. Daugherty, and R.P. Almeida. grafted trees of five of the rootstocks used in cultivars were grafted to some of the root- 2011. Grapevine genotype susceptibility to the test reported here. The incidence of stocks tested in this study, ‘Cape Fear’ trees Xylella fastidiosa does not predict vector trans- PBLS in the nongrafted rootstock trees in grafted to rootstock produced from ‘Curtis’ mission success. Environ. Entomol. 40:1192– this orchard after 10 years was similar to the seed did not have a lower incidence of 1199.

HORTSCIENCE VOL. 50(8) AUGUST 2015 1185 Redak, R.A., A.H. Purcell, J.R. Lopes, M.J. Blua, Sanderlin, R.S. and K.I. Heyderich-Alger. 2000. Sanderlin, R.S. and R.A. Melanson. 2008. Re- R.F. Mizel, and P.C. Anderson. 2004. The Evidence that Xylella fastidiosa can cause leaf duction of Xylella fastidiosa transmission biology of xylem fluid-feeding insect vectors scorch disease of pecan. Plant Dis. 84:1282– through pecan scion wood by hot-water treat- of Xylella fastidiosa and their relation to 1286. ment. Plant Dis. 92:1124–1126. disease epidemiology. Annu. Rev. Entomol. Sanderlin, R.S. and K.I. Heyderich-Alger. 2003. Sanderlin, R.S. and R.A. Melanson. 2010. Insect 49:243–270. Effects of pecan bacterial leaf scorch on growth transmission of Xylella fastidiosa to pecan. Sanderlin, R.S. 2005. Cultivar and seedling and yield components of cultivar Cape Fear. Plant Dis. 94:465–470. susceptibility to pecan bacterial leaf scorch Plant Dis. 87:259–262. Xu, X., H. Huang, and J. Lu. 2002. Investigating caused by Xylella fastidiosa and graft trans- Sanderlin, R.S. and R.A. Melanson. 2006. Trans- the Xylella fastidosa in Pierce’s disease re- mission of the pathogen. Plant Dis. 89:446– mission of Xylella fastidiosa through pecan sistant and susceptible grapevines. Proc. Fla. 449. rootstock. HortScience 41:1455–1456. State Hort. Soc. 115:105–108.

1186 HORTSCIENCE VOL. 50(8) AUGUST 2015