Vector Transmission Efficiency of Liberibacter by Bactericera Cockerelli (Hemiptera: Triozidae) in Zebra Chip Potato Disease: Ef

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ARTHROPODS IN RELATION TO PLANT DISEASE Vector Transmission Efﬁciency of Liberibacter by Bactericera cockerelli (Hemiptera: Triozidae) in Zebra Chip Potato Disease: Effects of Psyllid Life Stage and Inoculation Access Period

1,2 1 1,3 JEREMY L. BUCHMAN, VENKATESAN G. SENGODA, AND JOSEPH E. MUNYANEZA

J. Econ. Entomol. 104(5): 1486Ð1495 (2011); DOI: http://dx.doi.org/10.1603/EC11123 ABSTRACT Successful transmission of plant pathogens by insects depends on the vector inoculation efÞciency and how rapidly the insect can effectively transmit the pathogen to the host plant. The potato psyllid, Bactericera cockerelli (Sˇulc), has recently been found to transmit “Candidatus Liberib- acter solanacearum,” a bacterium associated with zebra chip (ZC), an emerging and economically important disease of potato in several parts of the world. Currently, little is known about the epidemiology of ZC and its vectorÕs inoculation capabilities. Studies were conducted in the Þeld and laboratory to 1) assess transmission efÞciency of potato psyllid nymphs and adults; 2) determine whether psyllid inoculation access period affects ZC incidence, severity, and potato yield; and 3) determine how fast the psyllid can transmit liberibacter to potato, leading to ZC development. Results showed that adult potato psyllids were highly efÞcient vectors of liberibacter that causes ZC and that nymphs were less efÞcient than adults at transmitting this bacterium. It was also determined that inoculation access period had little inßuence on overall ZC disease incidence, severity, and resulting yield loss. Moreover, results showed that exposure of a plant to 20 adult potato psyllids for a period as short as 1 h resulted in ZC symptom development. Furthermore, it was shown that a single adult potato psyllid was capable of inoculating liberibacter to potato within a period as short as 6 h, thereby inducing development of ZC. This information will help in developing effective management strategies for this serious potato disease.

KEY WORDS potato psyllid, liberibacter, inoculation efÞciency, potato, zebra chip

The potato psyllid, Bactericera cockerelli (Sˇulc) More recently, the potato psyllid has been shown to (Hemiptera: Triozidae), is a serious insect pest of be associated with zebra chip (ZC), an emerging and solanaceous crops, particularly potato (Solanum tu- economically important disease of potato in the south- berosum L.). The psyllid is thought to be native to the western United States, Mexico, Central America, and southwestern United States and northern Mexico, New Zealand (Munyaneza et al. 2007a,b, 2008; Lieft- where it also overwinters (Pletsch 1947, Wallis 1955). ing et al. 2008; Secor et al. 2009; Crosslin et al. 2010; The potato psyllid has a temperature-induced migra- Munyaneza 2010). The disease has been associated tion during the spring from overwintering sites into with a previously undescribed species of liberibacter northern potato producing regions of the United tentatively named “Candidatus Liberibacter so- States and southern Canada (Pletsch 1947, Wallis lanacearum” (syn. “Ca. L. psyllaurous”), transmitted 1955). This insect has historically been associated with by the potato psyllid (Hansen et al. 2008; Liefting et a condition called “psyllid yellows” of potatoes, char- al. 2008, 2009; Secor et al. 2009; Crosslin et al. 2010; acterized by leaf chlorosis and purpling, shortened Munyaneza 2010). The disease causes initial foliar internodes, the formation of aerial tubers, and reduc- chlorosis and subsequent purpling, shortened inter- tion of potato tuber yield (Richards and Blood 1933). nodes and production of aerial tubers, and leaf scorch- Psyllid yellows disease is thought to be associated with ing. These aboveground potato symptoms resemble feeding by psyllid nymphs (List 1925) and may be those associated with psyllid yellows and potato pur- caused by a toxin associated with the insect (Carter ple top diseases (Secor and Rivera-Varas 2004; Mun- 1939), though the actual etiology of the disease has yet yaneza et al. 2007a,b, 2008). The most characteristic to be determined (Sengoda et al. 2010). symptom of ZC is the production of dark ßecking concentrated in the perimedullary tissue within the potato tuber (Fig. 1A). This defect becomes more 1 Yakima Agricultural Research Laboratory, USDAÐARS, Wapato, pronounced when tubers are fried, producing regular WA 98951. striped patterns in latitudinal tuber slices (Fig. 1B) 2 Department of Entomology, Washington State University, Pull- man, WA 99164. and giving the disease its name of “zebra chip” (Mu- 3 Corresponding author, e-mail: [email protected]. nyaneza et al. 2007a,b, 2008; Secor et al. 2009; Crosslin October 2011 BUCHMAN ET AL.: EFFICIENCY OF B. cockerelli IN ZEBRA CHIP DISEASE 1487 et al. 2010; Miles et al. 2010). Chips or fries processed The individual plot under each cage measured 1.8 m from ZC-infected tubers are commercially unaccept- in length by 0.9 m in width and the ground was hilled able. Thus, the disease has caused millions of dollars in into a mound Ϸ0.3 m in height. The frame of each cage losses to the potato industry, often leading to aban- was formed by inserting both ends of 3.0-m-long Þ- donment of entire potato Þelds (Munyaneza et al. berglass poles (Geotek, Inc., Stewartville, MN) into 2007a,b, 2008; Secor et al. 2009; Crosslin et al. 2010). the ground at opposite ends of the plot, to form hoops Diseases caused by plant pathogens that require over the mounds. A third Þberglass pole was placed insect vectors for dissemination are typically managed halfway between the outside poles forming a central by controlling the vector population or by disrupting support. Insect proof fabric, Econet SF (USGR, Inc., the vectorÕs ability to acquire and inoculate the patho- Seattle, WA) 3.0 m in width and 4.6 m in length, was gen to and from hosts. This approach requires basic draped over the frame and the edges were buried in epidemiological knowledge about the vector and the the ground (Fig. 2A). The cages were arranged in pathogen and their interaction with the host. Infor- three rows of Þve cages each in a block; four blocks of mation on mechanisms of liberibacter transmission to cages were used, with a total of 60 cages. Each cage was potato by the potato psyllid is lacking. Development of spaced Ϸ1.5 m from the next one endwise and a spac- effective management strategies for ZC will not be ing of 0.9 m was placed between rows. Potato (Atlan- realized until transmission biology is better under- tic) plants in the cages were grown from certiÞed stood. The objectives of this research were to 1) assess disease-free potato seed (Atlantic) obtained from Th- the transmission efÞciency of potato psyllid nymphs aemert Farms (Quincy, WA). Six whole potato seed and adults; 2) determine the effect of inoculation tubers were planted per cage in late April 2008, with access period of single and multiple potato psyllid a spacing of 23 cm between seed tubers and planted adults under laboratory and Þeld conditions; and 3) at a depth of 13 cm. Fertilizer (Simplot Grower So- assess the impact of inoculation access duration on dis- lutions 16Ð16-16, Simplot Grower Solutions, Halsey, ease incidence and severity, and potato yield. OR) was side-dressed at the time of planting at a rate of 37.4 g per plot. Drip tape irrigation (Queen Gil, Intl. Burgas, Bulgaria) was used throughout the season to Materials and Methods water the potatoes. Experiments consisted of inoculating potato plants Two factors, insect life stage (nymphs or adults) and with “Ca. L. solanacearum” by exposing them to li- duration of plant exposure to psyllids, were examined beribacter-infective potato psyllids (i.e., carrying the to determine whether either affects incidence and bacterium) under controlled laboratory and Þeld con- severity of ZC disease in potatoes under Þeld condi- ditions and monitoring them for ZC symptom devel- tions. The experiment was a randomized complete opment. The potato psyllids used in the experiments block design, with 14 treatments in total. Treatments were from a liberibacter-infective colony that was were replicated four times each. Three psyllid life established and maintained at the USDAÐARS facility stage treatments were used: nymphs only, adults only, in Wapato, WA, with psyllids collected from ZC-in- and a mixed treatment of both nymphs and adults. Six fected experimental potato plots in Weslaco, TX, in plant exposure durations were also examined: 1, 3, 7, 2007. The insects were continuously reared for several 14, 30, and 60 d. Due to the difÞculty of keeping some generations on potato (ÔAtlanticÕ, a highly ZC-suscep- life stage treatments separate in the longer exposure tible cultivar) plants in BugDorm cages (Bioquip durations (14Ð60 d), the nymphs only and adults only Products, Rancho Dominguez, CA) in an insect rear- treatments were restricted to the three shortest ex- ing room maintained at 25 ЊC, 40% humidity, and a posure durations (1, 3, and 7 d), whereas the mixed photoperiod of 16:8 (L:D) h. nymphsÐadults treatment was subjected to all the Experimental Designs and Procedures. Effect of plant exposure durations (1Ð60 d). One cage in each Psyllid Life Stage on Liberibacter Transmission Efﬁ- block was left psyllid-free to serve as a control treat- ciency and Assessment of Inoculation Access Period Un- ment. Reproduction of psyllids occurred in the 30- and der Field Conditions. To assess the effect of potato 60-d plant exposure duration treatments. psyllid life stage transmission potential of liberibacter At the potato tuber initiation stage, psyllids were to potato and various inoculation access durations on released onto the leaves of each potato plant in the the production of ZC in potatoes, a Þeld cage trial was cage. This plant stage was selected to ensure tuber Þrst conducted at the USDAÐARS facility in Wapato, production and visual assessment of characteristic ZC WA, during summer 2008. Potato plants were grown in symptoms in tubers. In the nymphs or adults only small Þeld cages and exposed to liberibacter-infective treatments, 20 individuals were released on each plant. potato psyllids. The cages were used as enclosures to In the mixed nymphs and adults treatment, 10 indi- prevent escape of the potato psyllids released onto the viduals of each life stage were released onto each plants and to exclude other potato pests. The soil at the plant. Adults were collected from a liberibacter-in- site is sandy loam and was treated with preplant her- fective colony by using an insect aspirator, whereas bicides S-ethyl dipropylthicarbamate (Eptam, Gowan the nymphs (second to fourth instar) were carefully Co., Yuma, AZ) and trißuralin (Treßan, Dow Agro- removed from plant leaves in the colony and trans- sciences, LLC Calgary, AB, Canada) at label rates ferred to healthy plants in the Þeld cages by using an before planting for weed control. Additional weeding insect pin (size 0, Bioquip Products, Rancho Domin- was done by hand as needed throughout the season. guez, CA). At the end of each plant exposure duration, 1488 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 5

Fig. 1. Typical zebra chip disease symptoms in raw tubers (A) and fried chips (B). psyllids were eliminated from the test plants by spray- 2008). Percentage of infected tubers per plant was also ing them with an insecticide mixture of thiamethoxam estimated. No information regarding “Ca. L. so- (Actara, Syngenta, Greensboro, NC), acetamiprid lanacearum” was available at the time of this trial; thus, (Assail, DuPont, Tokyo, Japan) and spiromesifen no pathogen testing was performed either on the (Oberon, Bayer Cropscience, Research Triangle Park, plants or insects to conÞrm liberibacter infection. Dis- NC) at recommended label rates. This insecticide ease severity in tubers was measured for Þve medium- mixture was selected to ensure that all potato psyllid sized tubers (50Ð250 g) selected from each ZC-in- life stages are completely eliminated from the plants. fected plant. Using a Mandolin kitchen slicer (OXO, Tubers from potato plants in all the cages were Inc., Chambersburg, PA), a latitudinal slice (1 mm in individually hand-harvested in late July 2008 when the thickness) was taken 1 cm from the stem end of the potato plants were estimated to have reached maturity tuber. Also, a longitudinal slice (1 mm in thickness) (Webb et al. 1978) and visually assessed for ZC symp- was taken from the center of the tuber. Using a Frito- toms. A plant was considered diseased if at least one Lay chip defect severity scale of 0 (nonsymptomatic) tuber from the plant had typical ZC symptoms either to 5 (highly symptomatic), a visual score was assigned in raw or fried chip form (Munyaneza et al. 2007a,b, to each raw latitudinal tuber slice (Munyaneza et al.

Fig. 2. Enclosures used to conduct plant inoculation with liberibacter using potato psyllids in the Þeld (A) and laboratory (B). October 2011 BUCHMAN ET AL.: EFFICIENCY OF B. cockerelli IN ZEBRA CHIP DISEASE 1489

2008). The slices were then deep-fried in canola oil for 16, 20, or 24 h. Each inoculation access period trial with 3 min at 191ЊC by using a kitchen deep fryer (Rival, multiple insects was replicated with Þve or six plants. Inc., Boca Raton, FL) according to Munyaneza et al. A set of plants was not exposed to psyllids to serve as (2007a). After frying, visual scores (0Ð5) were as- controls and replicated the same number of times as signed to the fried chips as well. Scores were averaged the exposed plants. After each plant exposure period, across the tubers from each plant and the resulting psyllids were recollected from each plant and four score was averaged again across all the plants within psyllids were randomly selected and tested by poly- each cage. Uninfected tubers (tubers with both a raw merase chain reaction (PCR) assay to conÞrm the and fried score of zero) from infected plants were presence of “Ca. L. solanacearum.” For the single in- excluded in the severity calculations. Moreover, po- sect inoculation trials, the psyllids were released onto tato yield was estimated by counting the number of plants near the base of the stem and allowed access to tubers per plant and weighing individual tubers on a the plant for 6, 12, 24, and 72 h; unlike the multiple laboratory scale (model GT 2100, Ohaus Corp., Pine psyllid inoculation experiment, more time was used Brook, NJ). for single psyllids to increase their chance to effec- Inoculation Access Period Study Under Laboratory tively inoculate the plants with liberibacter. Inocula- Conditions. Based on the data from the above Þeld tion access periods for single psyllids were replicated inoculation study (see Results), we focused on further twice with nine or 10 plants each, in addition to one assessing inoculation access periods of Յ72 h and by control (psyllid-free) plant. The psyllids were recol- using adult potato psyllids rather than nymphs be- lected from each exposed plant and individually tested cause a single day inoculation access period had for the presence of “Ca. L. solanacearum” by PCR as proved to result in high incidence of ZC and nymphs described above. were less effective at inducing the disease. A series of After manual insect removal, the plants were laboratory-based individual potato plant inoculation treated in a fumigation chamber with 60-cL of methyl trials were conducted at the USDAÐARS facility in bromide for2htoeliminate potential remaining psyl- Wapato between December 2009 and February 2011. lids, their eggs, and other unwanted insects. After First, plants were individually exposed to 20 potato fumigation, the plants were uncovered and main- psyllids from a liberibacter-infective colony for se- tained in a psyllid-free greenhouse maintained at 24Ð lected periods of time to conÞrm the ZC symptom 28ЊC, 50 Ϯ 5% RH, and a photoperiod of 16:8 (L:D) h. development observations made in the Þeld disease The plants were visually monitored for development inoculation trial. The experiment was repeated twice. of ZC foliar symptoms. Approximately 75Ð90 d after In the second study, single insects were used to inoc- plant exposure to psyllids, tubers were collected from ulate individual plants and to determine the minimum each plant and assessed for ZC symptoms as described inoculation access period needed for a single psyllid to above. Similarly to the Þeld inoculation study, a plant induce ZC; two trials were conducted for this exper- was considered diseased if any tubers produced by the iment. Plants used in both single and multiple potato plant were symptomatic. For each plant, symptomatic psyllid inoculation access period experiments were tubers or associated stolon material (Li et al. 2009) grown from tissue culture-produced potato minitu- were collected and tested for liberibacter by PCR to bers (Atlantic or FL1867; FL1867 is a ZC-susceptible conÞrm infection. Frito-Lay proprietary variety) that were obtained Testing for Liberibacter. DNA Extraction for Plant from CSS Farms, Inc. (Colorado City, CO). The po- Material. Total DNA was extracted from healthy and tatoes were grown in 10.2-cm rectangular pots (Kord ZC-infected potato tissues by using cetyltrimethlyam- Products, Toronto, ON, Canada) in the greenhouse. monium bromide (CTAB) buffer extraction per Pas- The soil medium consisted of 86% sand, 13.4% peat trik and Maiss (2000) and Munyaneza et al. (2010a). moss, 0.5% Apex time release fertilizer (J. R. Simplot Four hundred milligrams of potato tissue (stem, pet- Co., Lathrop, CA), and 0.1% Micromax micronutrients ioles, and leaf tissues) was macerated with 1 ml of (Scotts Co., Marysville, OH). Again, potato plants extraction buffer (100 mM Tris-HCl, pH 8.0, 50 mM were at the tuber initiation stage when exposed to EDTA, 500 mM NaCl, and 10 mM mercaptoethanol) psyllids. Each plant was enclosed in a small hoop cage by using BioReba sample bags and a Homex 6 homog- made of 40-cm metallic ßag wires (GemplerÕs Inc., enizer (Bioreba, Reinach, Switzerland). Three hun- Madison, WI) inserted into the four corners of the pot dred milliliters of macerate was collected and mixed to create a frame over the plant (Fig. 2B). The frame with 80 ␮l of lysozyme (50 mg/ml in 10 mM Tris-HCl, was covered with 0.5- by 0.5-m piece of Econet SF pH 8.0, Sigma-Aldrich, St. Louis, MO) and incubated insect screen fabric (USGR, Inc., Seattle, WA), and for 30 min at 37ЊC. After incubation, 500 ␮l of CTAB the net was secured to the base of the pot with a buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM cloth-covered telephone cord (Oldphonesworks Inc., Tris-HCl, pH 8.0, and 0.2% mercaptoethanol) was Kingston, ON, Canada) (Fig. 2B). added to the homogenate, and the sample was incu- Adult potato psyllids were collected from a liberib- bated for 30 min at 65ЊC. The samples were maintained acter-infective colony and starved for 12 h before at room temperature (22ЊC) for 3 min, and 500 ␮lof being used in the inoculation experiments, to force ice-cold chloroform was added. Samples were vor- them to feed upon plant exposure. Twenty potato texed and then centrifuged at 14,000 rpm for 10 min. psyllids were released at the base of the potato plant The aqueous layer was then transferred to a new and allowed access to the plant for 0.5, 1, 2, 3, 4, 6, 8, microfuge tube containing 500 ␮l of isopropanol. The 1490 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 5 tube was placed on ice for 20 min to precipitate DNA. treatments were made using TukeyÕs student range DNA was recovered by centrifugation as described test. above. The pellet was washed with ice-cold 70% eth- Laboratory Inoculation Trials. The laboratory trials anol and centrifuged at 14,000 rpm for 2 min, and the were intended to conÞrm the Þeld observations and pellet was air-dried. The pellet was resuspended in 50 determine the minimum inoculation access periods ␮l of sterile water. needed by adult potato psyllids to successfully inoc- DNA Extraction for Psyllids. Total DNA was ex- ulate each plant and not to investigate differences in tracted from potato psyllids by using CTAB buffer exposure period length. Thus, no statistical analysis extraction (Zhang et al. 1998) but without grinding in was run on the treatments. In the single psyllid inoc- liquid nitrogen. Using a micropestle, individual psyl- ulations, any psyllid that tested negative for the pres- lids were ground in 1.5-ml sample tubes (Eppendorf ence of “Ca. L. solanacearum” and its corresponding North America, Hauppauge, NY) in 500 ␮l of CTAB plant was not included in the data tables. buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, and 0.2% mercaptoethanol). The Results samples were incubated for 30 min at 65ЊC. The samples were maintained at room temperature (22ЊC) for Field Inoculation Trials. ZC disease symptoms de- 3 min, and then 500 ␮l of ice-cold chloroform was veloped in all treatments in which plants were exposed added. Samples were vortexed and then centrifuged at to psyllids of any life stage, whereas the unexposed 14,000 rpm for 10 min. The aqueous layer was then controls showed no symptoms of the disease. ZC in- transferred to a new microfuge tube containing 0.6 cidence per plant was 100% in all of the treatments in volume of isopropanol and 1 ␮l of glycogen. The tube which plants were exposed to adult psyllids (Tables 1 was placed on ice for 20 min to precipitate DNA. DNA and 2). This lack of variation prevented any mean- was recovered by centrifugation as described above. ingful statistical analysis. Disease incidence in tubers The pellet was washed with ice-cold 70% ethanol and showed more variation but any potatoes exposed to centrifuged at 14,000 rpm for 2 min, and the pellet was psyllid adults showed very high rates of infection and air-dried. The pellet was resuspended in 100 ␮lof disease symptom expression. No interaction was found sterile water. between the psyllid life stage and inoculation access periods (F ϭ 0.6; df ϭ 4, 24; P ϭ 0.65), and there was Detection of “Ca. L. solanacearum” With PCR. De- no signiÞcant difference between relatively short in- tection of “Ca. L. solanacearum” was performed with oculation access periods of a week or less (F ϭ 0.4; df ϭ primer pair OA2/OI2c (Crosslin and Munyaneza 2009; 4, 24; P ϭ 0.65). Psyllid life stage treatment was found Liefting et al. 2008, 2009). In brief, PCR was performed to affect incidence in tubers (F ϭ 63.0; df ϭ 2, 24; P Ͻ with 50-␮l reactions containing 1ϫ Green GoTaq re- 0.0001). Potatoes exposed to the nymphs only treat- action buffer (Promega, Madison, WI), 100 ␮M ␮ ment had a signiÞcantly lower ZC incidence in tubers dNTPs, 20 pmol of each primer, 2 l of DNA extracts, than incidence in plants exposed to adults only and and1UofGoTaq Polymerase (Promega). The PCR Њ mixed adults and nymphs treatments (Table 1). These cycling conditions were 94 C for 2 min followed by 40 data support the trends seen in the plant incidence cycles at 94ЊC for 30 s, 65ЊC for 30 s, and 72ЊC for 60 s Њ data. ZC incidence in tubers was very high among the followed by a Þnal incubation at 72 C for 5 min (MJ mixed adult-nymph treatments across all inoculation Research, Watertown, MA). Ten ml of the reactions access periods but no signiÞcant difference was found were analyzed by electrophoresis in 1.5% agarose gels, among the exposure periods (F ϭ 1.2; df ϭ 5, 15; P ϭ stained with ethidium bromide, and observed under 0.37) (Table 2). UV light. Presence of the predicted 1,168-bp 16S In tubers displaying ZC symptoms, frying tended to rDNA band indicated samples were positive for “Ca. L. increase the overall darkness and hence disease se- solanacearum.” verity (Tables 1 and 2). No interaction was found Statistical Analysis. Field Inoculation Trial. The data between psyllid life stage and inoculation access pe- were analyzed as two separate analysis of variance riods of a week or less either in raw tuber (F ϭ 0.5; df ϭ (ANOVA) comparisons. First, to measure life stage 4, 24; P ϭ 0.73) or in fried tuber disease severity (F ϭ and short inoculation access period impact on ZC 0.6; df ϭ 4, 24; P ϭ 0.64). Psyllid life stage was found development, the treatments were analyzed asa3by to affect disease severity in raw tubers (F ϭ 4.1; df ϭ 3 factorial design (life stage: nymphs only, adults only 2, 24; P ϭ 0.029); severity score was lowest in the and mixed nymphs and adults ϫ short plant exposure nymphs only treatment. However, psyllid life stage did duration: 1, 2, and 7 d). Second, to measure the effects not affect ZC severity in fried tubers (F ϭ 1.8; df ϭ 2, of all inoculation access periods, a one-way ANOVA 24; P ϭ 0.19). Plant exposure to psyllids duration did was used to analyze incidence and yield across all six not affect ZC severity in raw tuber (F ϭ 1.4; df ϭ 2, 24; plant exposure durations, limiting the analysis to the P ϭ 0.28) or in fried tubers (F ϭ 0.47; df ϭ 2, 24; P ϭ mixed life stage treatment. The control treatment was 0.63). In analyzing all inoculation access periods in- excluded in the analyses of incidence and severity, volving mixed nymphÐadult treatments, there was a because both measurements were found to be nega- weak (but signiÞcant) trend for increasing disease tive for the disease (as expected) in all control plants. severity associated with plant exposure duration both The analyses were done using PROC MIXED in SAS for raw tubers (F ϭ 4.9; df ϭ 5, 15; P ϭ 0.0076) and fried (SAS Institute 2003). Pairwise comparisons among tubers (F ϭ 3.0; df ϭ 5, 15; P ϭ 0.043) (Table 2). October 2011 BUCHMAN ET AL.: EFFICIENCY OF B. cockerelli IN ZEBRA CHIP DISEASE 1491

Table 1. Effects of different potato psyllid life stages and inoculation access period of a week or less on zebra chip potato disease incidence and severity

Life stage Inoculation access Zebra chip incidence Zebra chip incidence Zebra chip severity in Zebra chip severity in treatment period (d)a in plants (%) in tubers (%) raw chips (avg score) fried chips (avg score) Main effect meansb Nymphs only 68.1 31.0 Ϯ 4.7a 2.2 Ϯ 0.3a 3.2 Ϯ 0.3 Adults only 100.0 95.5 Ϯ 4.7b 3.3 Ϯ 0.3b 3.9 Ϯ 0.3 Nymphs and adults 100.0 95.6 Ϯ 4.7b 3.2 Ϯ 0.3ab 3.6 Ϯ 0.3 1 90.3 71.2 Ϯ 4.7 2.7 Ϯ 0.3 3.6 Ϯ 0.3 3 87.5 77.4 Ϯ 4.7 3.3 Ϯ 0.3 3.7 Ϯ 0.3 7 90.3 73.5 Ϯ 4.7 2.8 Ϯ 0.3 3.4 Ϯ 0.3 Interaction meansb Nymphs only 1 70.8 19.6 Ϯ 8.1 1.7 Ϯ 0.5 3.3 Ϯ 0.5 3 62.5 38.1 Ϯ 8.1 3.1 Ϯ 0.5 3.7 Ϯ 0.5 7 70.8 35.3 Ϯ 8.1 1.9 Ϯ 0.5 2.5 Ϯ 0.5 Adults only 1 100.0 96.8 Ϯ 8.1 3.2 Ϯ 0.5 3.9 Ϯ 0.5 3 100.0 97.0 Ϯ 8.1 3.4 Ϯ 0.5 3.8 Ϯ 0.5 7 100.0 92.5 Ϯ 8.1 3.4 Ϯ 0.5 4.0 Ϯ 0.5 Nymphs and adults 1 100.0 97.1 Ϯ 8.1 3.1 Ϯ 0.5 3.5 Ϯ 0.5 3 100.0 97.0 Ϯ 8.1 3.5 Ϯ 0.5 3.7 Ϯ 0.5 7 100.0 92.7 Ϯ 8.1 3.1 Ϯ 0.5 3.7 Ϯ 0.5

a Control plants (not exposed to psyllids) were uniformly zebra chip-free at the end of the experiments. b Means followed by same letters within columns are not signiÞcantly different at P ϭ 0.05 (ANOVA; TukeyÕs student range test).

The yield data related to the different psyllid life affected by plant exposure duration (F ϭ 18.6; df ϭ 6, stage treatments over the short inoculation access 18; P Ͻ 0.0001), primarily because of the higher yields durations of a week or less are summarized in Table 3. in the control plants. Numbers of tubers per plant No interaction between psyllid life stage and inocu- were not affected by plant exposure duration (F ϭ 1.0; lation access period was found for any of the Þve yield df ϭ 6, 18; P ϭ 0.45). A statistically signiÞcant decline measurements analyzed (P Ͼ 0.55 for all measurement in average tuber size was detected as plant exposure categories). Tuber yield per plant was found to be durations increased (F ϭ 3.8; df ϭ 6, 18; P ϭ 0.013). affected by psyllid life stage (F ϭ 37.3; df ϭ 2, 24; P Ͻ Laboratory Inoculation Trials. Liberibacter infec- 0.0001). Yield was higher in the nymphs only treat- tion rate in psyllids used in the laboratory inoculation ment than the adults only and mixed nymphÐadult trials was estimated between 90 and 100%, by using treatments. Plant exposure duration did not affect PCR assays. The results for the multiple psyllid and tuber yield per plant (F ϭ 0.3; df ϭ 2, 24; P ϭ 0.77). The single inoculation trials are summarized in Table 5. average number of tubers per plant was found to be The data show that exposure of potato plants to 20 signiÞcantly higher in the nymph only treatment com- adult potato psyllids for a period of time as short as 1 h pared with the other two treatments (F ϭ 14.9; df ϭ resulted in ZC symptom development. The results for 2, 24; P Ͻ 0.0001), and again there was no signiÞcant the single insect inoculation trials show that exposure effect associated with inoculation access period (F ϭ of plants to single liberibacter-infective potato psyllids 1.8; df ϭ 2, 24; P ϭ 0.19). Tuber size was found to be resulted in development of ZC in as short as 6 h. affected by psyllid life stage (F ϭ 5.5; df ϭ 2, 24; P ϭ Conventional PCR testing of ZC-infected plants 0.011). Larger tubers were produced in the nymphs conÞrmed presence of “Ca. L. solanacearum” in the only treatment than the other two treatments. Tuber inoculated plants. However, variation was observed size was not affected by plant exposure duration (F ϭ between the PCR results from plant samples and visual 0.7; df ϭ 2, 24; P ϭ 0.53). Means for the yield data symptom assessment in both the multiple and single related to long inoculation access periods in the mixed psyllid inoculation trials. In the multiple psyllid inoc- nymphÐadult treatments and the control are summa- ulation trials, 38 of 49 (77.5%) visually identiÞed ZC- rized in Table 4. Per plant yield was found to be infected plants tested positive for “Ca. L. so-

Table 2. Effects of inoculation access periods up to 60 d with a mixture of potato psyllid nymphs and adults on zebra chip potato disease incidence and severity

Inoculation access Zebra chip incidence Zebra chip incidence Zebra chip severity Zebra chip severity in period (d)a,b in plants (%) in tubers (%) in raw chips (avg score) fried chips (avg score) 1 100.0 97.1 Ϯ 2.1 3.1 Ϯ 0.2a 3.5 Ϯ 0.2a 3 100.0 97.0 Ϯ 2.1 3.5 Ϯ 0.2ab 3.7 Ϯ 0.2ab 7 100.0 92.7 Ϯ 2.1 3.1 Ϯ 0.2a 3.7 Ϯ 0.2ab 14 100.0 97.1 Ϯ 2.1 3.2 Ϯ 0.2ab 3.8 Ϯ 0.2ab 30 100.0 92.7 Ϯ 2.1 3.8 Ϯ 0.2ab 4.1 Ϯ 0.2ab 60 100.0 97.4 Ϯ 2.1 3.9 Ϯ 0.2b 4.3 Ϯ 0.2b

Table 3. Effects of different potato psyllid life stages and inoculation access period of a week or less on potato tuber yield

Life stage Inoculation access Avg tuber yield Avg no. tubers Avg tuber treatment period (d) per plant (g) per plant size (g) Main effect meansa Nymphs only 1,886.3 Ϯ 67.2a 23.9 Ϯ 1.0a 99.8 Ϯ 5.8a Adults only 1,210.3 Ϯ 67.2b 18.1 Ϯ 1.0b 76.9 Ϯ 5.8b Nymphs and adults 1,144.8 Ϯ 67.2b 17.0 Ϯ 1.0b 75.9 Ϯ 5.8b 1 1,441.4 Ϯ 67.2 20.5 Ϯ 1.0 80.0 Ϯ 5.8 3 1,375.1 Ϯ 67.2 18.2 Ϯ 1.0 89.1 Ϯ 5.8 7 1,424.8 Ϯ 67.2 20.3 Ϯ 1.0 83.5 Ϯ 5.8 Interaction means1 Nymphs only 1 1,926.6 Ϯ 116.5 24.2 Ϯ 1.7 96.1 Ϯ 10.0 3 1,867.6 Ϯ 116.5 22.1 Ϯ 1.7 105.5 Ϯ 10.0 7 1,864.6 Ϯ 116.5 25.3 Ϯ 1.7 97.7 Ϯ 10.0 Adults only 1 1,175.1 Ϯ 116.5 18.1 Ϯ 1.7 68.9 Ϯ 10.0 3 1,273.4 Ϯ 116.5 17.7 Ϯ 1.7 91.4 Ϯ 10.0 7 1,182.3 Ϯ 116.5 18.6 Ϯ 1.7 70.4 Ϯ 10.0 Nymphs and adults 1 1,222.6 Ϯ 116.5 19.2 Ϯ 1.7 74.8 Ϯ 10.0 3 984.4 Ϯ 116.5 14.7 Ϯ 1.7 70.5 Ϯ 10.0 7 1,224.4 Ϯ 116.5 17.1 Ϯ 1.7 82.3 Ϯ 10.0

a Means followed by same letters within columns are not signiÞcantly different at P ϭ 0.05 (ANOVA; TukeyÕs student range test). lanacearum,” whereas in the single insect inoculation latent period may not play a major role in liberibacter trials, only 17 of 33 (51.5%) visually identiÞed ZC- transmission by nymphs produced by psyllids carrying infected plants tested positive for the bacterium. the bacterium. In addition, potato psyllid nymphs, due to their limited mobility, are likely less effective in spreading ZC than adults within local or distant potato Discussion Þelds. However, disease spread may be enhanced by Here, we determined that both nymphs and adults infective nymph hatches and the subsequent spread of of the potato psyllid effectively inoculated liberibacter potato psyllid adults developed on the infected plants. to potato, leading to ZC development in the Þeld cage Every treatment with potato psyllid adults showed inoculation trials. Under Þeld cage conditions, both very high rates of ZC infection for both plants and potato psyllid nymphs and adults were able to induce tubers. This suggests that adults are highly competent development of ZC in the exposed potato plants with vectors of ZC liberibacter. Combined with their high an inoculation access period as short as 1 d. The lower motility, potato psyllid adults probably represent the ZC disease incidence observed both in plants and highest threat for the spread of ZC, local or long tubers in the treatments with only nymphs may indi- distance. cate a reduced effectiveness at liberibacter inocula- Results of liberibacter inoculation studies under tion by potato psyllid nymphs compared with adults. laboratory conditions with multiple and single psyllids Similarly, the results suggested that psyllid nymphs conÞrmed the capability of potato psyllids to effec- had a lower impact on overall ZC severity in raw tively inoculate the bacterium to potato and lead to ZC tubers than adults. The reasons behind this reduced observed in the Þeld trials after the inoculation access ZC disease transmission and severity by nymphs are period of a single day. In addition, results showed that not clear but may be due to suspected differences in these insects were able to induce the potato disease feeding behavior or feeding sites between potato psyl- after even much shorter inoculation access durations. lid nymphs and adults. It is also possible that incidence Groups of 20 potato psyllids per plant successfully and titer of this bacterium in different psyllid life transmitted liberibacter to potato that caused ZC after stages may differ, affecting pathogen inoculation. It an inoculation access period of 1 h, whereas it took has been shown that liberibacter is vertically trans- Ϸ6-h inoculation access period for a single psyllid per mitted in the potato psyllid (Hansen et al. 2008), so plant to do the same. Multiple infective vectors on a

Table 4. Effects of inoculation access periods up to 60 d with a mixture of potato psyllid nymphs and adults on potato tuber yield

Life stage Inoculation access Avg tuber yield Avg no. tubers Avg tuber treatmenta period (d) per plant (g) per plant size (g) Control 0 1,831.2 Ϯ 77.7a 21.5 Ϯ 2.1 96.8 Ϯ 7.0a Nymphs and adults 1 1,222.6 Ϯ 77.7bc 19.2 Ϯ 2.1 74.8 Ϯ 7.0ab 3 984.4 Ϯ 77.7bc 14.8 Ϯ 2.1 70.5 Ϯ 7.0ab 7 1,227.4 Ϯ 77.7b 17.1 Ϯ 2.1 82.3 Ϯ 7.0ab 14 961.7 Ϯ 77.7bc 17.1 Ϯ 2.1 71.9 Ϯ 7.0ab 30 920.7 Ϯ 77.7bc 18.1 Ϯ 2.1 58.1 Ϯ 7.0b 60 861.2 Ϯ 77.7c 16.9 Ϯ 2.1 57.8 Ϯ 7.0b

a Means followed by same letters within columns are not signiÞcantly different at P ϭ 0.05 (ANOVA; TukeyÕs student range test). October 2011 BUCHMAN ET AL.: EFFICIENCY OF B. cockerelli IN ZEBRA CHIP DISEASE 1493

Table 5. Effects of different inoculation access periods with the yield differences observed in tuber yield, number, multiple or single psyllid adults on zebra chip potato disease inci- and size of plants exposed to nymphs over a 7-d in- dence oculation access period are probably due to reduced Total no. No. plants ZC infection of plants and tubers compared with any Inoculation access potato plants exhibiting zebra treatments with adults rather than any effect of direct period (h) exposed to chip disease feeding by the nymphs themselves. Any inoculation psyllids symptoms access duration resulted in a substantial reduction in Multiple psyllid inoculation yield per plant compared with the unexposed control 0110plants. Once plants were exposed to psyllids, the 0.5 6 0 1112length of exposure did not seem to affect the overall 263yield of each plant. This observation suggests that once 365the plant is inoculated with liberibacter, substantial 41110yield reduction in the plant will result. In addition, 665 8119reproduction of psyllids occurred in the 30- and 60-d 16 5 5 inoculation access period treatments. Regardless of 20 5 5 the presence of nymphs and the subsequent yield 24 5 5 reduction due to their feeding, the longer inoculation Single psyllid inoculation 0190access durations did not signiÞcantly reduce yield 6199more than the shortest ones, suggesting that the psyl- 12 19 5 lids have little additional impact on yield loss once a 24 19 4 plant is infected with liberibacter. In contrast, the 72 19 15 average tuber size decreased gradually as inoculation Twenty and one liberibacter-infective potato psyllids per potato access durations grew longer, and there was overlap plant were used in the multiple and single psyllid inoculation exper- with the control plants. Tuber size is a Þner measure- iments, respectively. ment of yield loss than whole plant yield and may reßect the more subtle physiological changes occur- ring in the plant after ZC infection. single plant would greatly increase the likelihood of Although information on liberibacter involvement successful inoculation. However, this short inocula- in ZC was nonexistent at the time the ZC Þeld inoc- tion access period for both multiple and single psyllids ulation trials were conducted and no testing for this represents a substantial challenge for growers in con- bacterium was performed, conventional PCR testing trolling the psyllid vector of ZC liberibacter. Just a few of ZC-infected plants conÞrmed the presence of “Ca. infective-psyllids feeding on potato for short periods L. solanacearum” in plants inoculated during the lab- could be responsible for substantial spread of the dis- oratory trials. The discrepancies in visual assessment ease within a potato Þeld or region. Conventional of ZC symptoms and PCR testing results observed pesticides may have limited direct disease control as during this study may be due to low titer of the bac- they may not kill the potato psyllid fast enough to terium in the inoculated plants, especially when single prevent liberibacter transmission, although they may insects were used. Quantitative real-time PCR detec- be useful for reducing the overall population of psyl- tion for “Ca. L. solanacearum” in ZC-infected potato lids. The most valuable and effective strategies to man- plant material has shown this bacterium to have an age ZC would probably be those that discourage vec- uneven distribution in planta (Li et al. 2009), often tor feeding, such as use of plants that are resistant to making PCR detection of this bacterium in potato psyllid feeding or non preference by the psyllid. Un- plants and tubers inconsistent. The titer of this liberib- fortunately, no potato variety has so far been shown to acter species in ZC-infected potato plants also may be exhibit sufÞcient resistance or tolerance to ZC or po- inßuenced by initial bacterium inoculum, inocula- tato psyllid (Munyaneza et al. 2010b). However, some tion site, and length of multiplication time since conventional and biorational pesticides, including inoculation, which may explain the relatively low plant and mineral oils and kaolin, have shown some liberibacter detection rate (51.5%) observed during substantial deterrence and repellency to potato psyllid the laboratory plant inoculation trials with single feeding and oviposition (Gharalari et al. 2009, Yang et potato psyllid adults compared with the detection al. 2010, Butler et al. 2011, Peng et al. 2011) and could rate for multiple psyllid inoculations, which was be useful tools in integrated pest management pro- estimated at 77.5%. grams to manage ZC and its psyllid vector. In summary, we have shown that although both potato Potato tuber yield has previously been shown to be psyllid nymphs and adults can effectively inoculate li- greatly reduced in ZC-infected potatoes (Munyaneza beribacter to potato, leading to ZC development, there et al. 2008). Psyllid yellows-affected potatoes also are differences in the inoculation capabilities and result- show a steep reduction in tuber yield due to the ing ZC symptom expression caused by nymphs com- continued presence of potato psyllid nymphs (Schall pared with adults. Adult potato psyllids are highly 1938), although psyllid yellows-affected plants will efÞcient vectors of ZC liberibacter and a single liberib- undergo a recovery of symptoms if the nymphs are acter-infective adult psyllid can effectively induce the removed from the plants (Sengoda et al. 2010). In the disease after a relatively short inoculation access period. current study, overall potato yield was reduced, and It also seems that short as well as long inoculation access 1494 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 5 durations of multiple or single psyllids have similar im- Munyaneza, J. E. 2010. Psyllids as vectors of emerging bac- pact on the eventual disease severity in potato tubers and terial diseases of annual crops. Southwest. Entomol. 35: yield loss due to ZC infection. This information on potato 417Ð477. psyllid inoculation of liberibacter to potato, coupled with Munyaneza, J. E., J. M. Crosslin, and J. E. Upton. 2007a. effective psyllid monitoring, can assist growers in making Association of Bactericera cockerelli (Homoptera: Psylli- effective management decisions to substantially reduce dae) with “zebra chip”, a new potato disease in south- the impact of this serious potato disease. western United States and Mexico. J. Econ. Entomol. 100: 656Ð663. Munyaneza, J. E., J. A. Goolsby, J. M. Crosslin, and J. E. Acknowledgments Upton. 2007b. Further evidence that zebra chip potato disease in the lower Rio Grande Valley of Texas is asso- We thank Blaine Heilman, Millie Heidt, Tonja Fisher, Dan ciated with Bactericera cockerelli. Subtrop. Plant Sci. 59: Hallauer, Jeff Upton, Jerry Gefre, and Andy Cruz for invalu- 30Ð37. able technical assistance. We are also very grateful to Dave Munyaneza, J. E., J. L. Buchman, J. E. Upton, J. A. Goolsby, Horton for help with statistical analysis. Financial support for J. M. Crosslin, G. Bester, G. P. Miles, and V. G. Sengoda. this research was partially provided by Frito-Lay, Inc.; 2008. Impact of different potato psyllid populations on USDAÐARS State Partnership Potato Program; Texas De- zebra chip disease incidence, severity, and potato yield. partment of Agriculture; USDAÐRisk Avoidance and Mitiga- Subtrop. Plant Sci. 60: 27Ð37. tion (2009-51101-05892) and USDAÐSpecialty Crop Re- Munyaneza, J. E., T. W. Fisher, V. G. Sengoda, S. F. Garc- search Initiative (2009-51181-20176). zynski, A. Nissinen, and A. Lemmetty. 2010a. Associa- tion of “Candidatus Liberibacter solanacearum” with the References Cited psyllid, Trioza apicalis (Hemiptera: Triozidae) in Europe. J. Econ. Entomol. 103: 1060Ð1070. Butler, C. D., F. R. Byrne, M. L. Keremane, R. F. Lee, and Munyaneza, J. E., J. L. Buchman, V. G. Sengoda, T. W. J. T. Trumble. 2011. Effects of insecticides on behavior Fisher, G. Bester, R. Hoopes, C. Miller, R. Novy, P. Van of adult Bactericera cockerelli (Hemiptera: Triozidae) and Hest, and J. Nordgaard. 2010b. Potato variety screen- transmission of Candidatus Liberibacter psyllaurous. J. ing trial for zebra chip resistance under controlled Þeld Econ. Entomol. 104: 586Ð594. cage conditions, pp. 200Ð203. In Proceedings, 2010 An- Carter, W. 1939. Injuries to plants caused by insect toxins. nual Zebra Chip Reporting Session, 7Ð10 November Bot. Rev. 5: 273Ð326. 2010, Dallas, TX. Crosslin, J. M., and J. E. Munyaneza. 2009. Evidence that the Pastrik, K. H., and E. Maiss. 2000. Detection of Ralstonia zebra chip disease and the putative causal agent can be solanacearum in potato tubers by polymerase chain re- maintained in potatoes by grafting and in vitro. Am. J. Potato Res. 86: 183Ð187. action. J. Phytopathol. 148: 619Ð626. Crosslin, J. M., J. E. Munyaneza, J. K. Brown, and L. W. Peng, L., J. T. Trumble, J. E. Munyaneza, and T.-X. Liu. 2011. Liefting. 2010. Potato zebra chip disease: a phytopatho- Repellency of a kaolin particle Þlm to potato psyllid, logical tale. Online. Plant Health Program. doi: http:// Bactericera cockerelli (Hemiptera: Psyllidae), on tomato dx.doi.org/10.1094/PHP-2010-0317-01-RV. under laboratory and Þeld conditions. Pest Manag. Gharalari, A. H., C. Nansen, D. S. Lawson, J. Gilley, J. E. Sci. 67: 815Ð824. Munyaneza, and K. Vaughn. 2009. Knockdown mortal- Pletsch, D. J. 1947. The potato psyllid Paratrioza cockerelli ity, repellency, and residual effects of insecticides for (Sulc) its biology and control. Mont. Agric. Exp. Stn. Bull. control of adult Bactericera cockerelli (Hemiptera: Psyl- 446. lidae). J. Econ. Entomol. 102: 1032Ð1038. Richards, B. L., and H. L. Blood. 1933. Psyllid yellows of the Hansen, A. K., J. T. Trumble, R. Stouthamer, and T. D. Paine. potato. J. Agric. Res. 46: 189Ð216. 2008. A new huanglongbing species, ÔCandidatus Li- SAS Institute. 2003. SAS userÕs guide: statistics, version 9.1. beribacter psyllaurousÕ found to infect tomato and potato, SAS Institute, Cary, NC. is vectored by the psyllid Bactericera cockerelli (Sulc). Schall, L. A. 1938. Some factors affecting the symptoms of Appl. Environ. Microbiol. 74: 5862Ð5865. the psyllid yellows disease of potatoes. Am. Potato J. 15: Li, W., J. A. Abad, R. D. French-Monar, J. Rascoe, A. Wen, 193Ð206. N. C. Gudmestad, G. A. Secor, I. M. Lee, Y. Duan, and L. Secor, G. A., and V. Rivera-Varas. 2004. Emerging diseases Levy. 2009. Multiplex real-time PCR for detection, iden- of cultivated potato and their impact on Latin America. tiÞcation and quantiÞcation of ÔCandidatus Liberibacter Rev. Latinoamer. Papa 1: 1Ð8 (Suppl.) solanacearumÕ in potato plants with zebra chip. J. Micro- Secor, G. A., V. Rivera-Varas, J. A. Abad, I.-M. Lee, G.R.G. biol. Methods 78: 59Ð65. Clover, L. W. Liefting, X. Li, and S. H. De Boer. 2009. Liefting, L. W., Z. C. Rez-Egusquiza, G.R.G. Clover, and Association of ÔCandidatus Liberibacter solanacearumÕ J.A.D. Anderson. 2008. A new ÔCandidatus LiberibacterÕ with zebra chip disease of potato established by graft and species in Solanum tuberosum in New Zealand. Plant Dis. 92: 1474. psyllid transmission, electron microscopy, and PCR. Plant Liefting, L. W., B. S. Weir, S. R. Pennycook, and G.R.G. Dis. 93: 574Ð583. Clover. 2009. ÔCandidatus Liberibacter solanacearumÕ, Sengoda, V. G., J. E. Munyaneza, J. M. Crosslin, J. L. Buch- associated with plants in the family Solanaceae. Int. J. man, and H. R. Pappu. 2010. Phenotypic and etiological Syst. Evol. Microbiol. 59: 2274Ð2276. differences between psyllid yellows and zebra chip dis- List, G. M. 1925. The tomato psyllid, Paratrioza cockerelli eases of potato. Am. J. Potato Res. 87: 41Ð49. Sulc. Colo. State Entomol. Circ. 47. Wallis, R. L. 1955. Ecological studies on the potato psyllid as Miles, G. P., M. A. Samuel, J. Chen, E. L. Civerolo, and J. E. a pest of potatoes. USDA Tech. Bull. 1107: 1Ð24. Munyaneza. 2010. Evidence that cell death is associated Webb, R. E., D. R. Wilson, J. R. Shumaker, B. Graves, M. R. with zebra chip disease in potato tubers. Am. J. Potato Henniger, J. Watts, J. A. Frank, and H. J. Murphy. 1978. Res. 87: 337Ð349. Atlantic: a new potato variety with high solids, good October 2011 BUCHMAN ET AL.: EFFICIENCY OF B. cockerelli IN ZEBRA CHIP DISEASE 1495

processing quality, and resistance to pests. Am. Potato J. Zhang, Y. P., J. K. Uyemoto, and B. C. Kirkpatrick. 1998. A 55: 141Ð145. small-scale procedure for extracting nucleic acids from Yang, X.-B., Y.-M. Zhang, L. Hau, L.-N. Peng, J. E. Munya- woody plants infected with various phytopathogens for neza, and T.-X. Liu. 2010. Repellency of selected biora- PCR assay. J. Virol. Methods 71: 45Ð50. tional insecticides to potato psyllid, Bactericera cockerelli (Hemiptera: Psyllidae). Crop Prot. 29: 1329Ð1324. Received 14 April 2011; accepted 8 July 2011.