SAMPLING AND BIOSTATISTICS Dispersion Patterns and Sampling Plans for Diaphorina citri (Hemiptera: Psyllidae) in Citrus 1 2 3 MAMOUDOU SE´ TAMOU, DANIEL FLORES, J. VICTOR FRENCH, AND DAVID G. HALL Texas A&M University Kingsville, Citrus Center, Weslaco, TX 78596 J. Econ. Entomol. 101(4): 1478Ð1487 (2008) ABSTRACT The abundance and spatial dispersion of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) were studied in 34 grapefruit (Citrus paradisi Macfad.) and six sweet orange [Citrus sinensis (L.) Osbeck] orchards from March to August 2006 when the pest is more abundant in southern Texas. Although ßush shoot infestation levels did not vary with host plant species, densities of D. citri eggs, nymphs, and adults were signiÞcantly higher on sweet orange than on grapefruit. D. citri immatures also were found in signiÞcantly higher numbers in the southeastern quadrant of trees than other parts of the canopy. The spatial distribution of D. citri nymphs and adults was analyzed using IowaÕs patchiness regression and TaylorÕs power law. TaylorÕs power law Þtted the data better than IowaÕs model. Based on both regression models, the Þeld dispersion patterns of D. citri nymphs and adults were aggregated among ßush shoots in individual trees as indicated by the regression slopes that were signiÞcantly Ͼ1. For the average density of each life stage obtained during our surveys, the minimum number of ßush shoots per tree needed to estimate D. citri densities varied from eight for eggs to four ßush shoots for adults. Projections indicated that a sampling plan consisting of 10 trees and eight ßush shoots per tree would provide density estimates of the three developmental stages of D. citri acceptable enough for population studies and management decisions. A presenceÐabsence sampling plan with a Þxed precision level was developed and can be used to provide a quick estimation of D. citri populations in citrus orchards. KEY WORDS Diaphorina citri, dispersion, sampling units, sample size The psyllid Diaphorina citri Kuwayama (Hemiptera: plants where direct feeding damage by the pest can Psyllidae), one of the vectors of Huanglongbing or cause reduced ßush growth (Michaud 2004) and citrus greening disease, has invaded two areas in the sometimes young shoot death. However, the recent United States over the past decade, Florida and Texas discovery of citrus greening disease and its phloem- (French et al. 2001, Halbert and Manjunath 2004). limited, nonculturable causal agent Candidatus Citrus greening disease was found in Florida during Liberibacter asiaticus in Florida (Halbert 2005) has 2005 (Halbert 2005), but it has not yet been reported dramatically increased the pest status of D. citri and in Texas. Citrus greening is probably the most impor- the threat posed by this disease to the U.S. citrus tant disease of citrus in the world (Aubert 1990, da industry. In a recent statewide survey in Texas, da Grac¸a 1991, Bove´ 2006). The presence of the disease Grac¸a et al. (2006) reported D. citri in 40% of the in Florida is threatening the proÞtability and sustain- counties where citrus was found throughout the state, ability of this stateÕs citrus industry. Citrus is an im- with all three counties where commercial citrus is portant agricultural industry in the United States, with grown being infested. The rapid spread of D. citri in Ϸ500,000 ha in citrus orchards primarily located in the Texas is not surprising given its high reproductive states of Florida, California, Texas, and Arizona, and an potential (Mead 1976, Liu and Tsai 2000), the favor- annual on-tree value of $1.3 billion (USDAÐNASS able climatic conditions and availability of its host 2005). plants in the state. D. citri infests leaves and ßush In the absence of the bacterial causal agents of citrus shoots of its host plants, all of which are restricted to greening disease, D. citri does not pose a major threat the Rutaceae family, including Citrus spp. (Aubert except for nursery and newly established orchard 1990). Eggs are laid exclusively on new ßush shoots (Hall and Albrigo 2007) of the host plant, and both 1 Corresponding author, e-mail: [email protected]. nymphs and adults preferentially feed on these 2 USDAÐAPHISÐPPQÐCPHST, Pest Detection Diagnostics and ßushes. Management Laboratory 22675 N. MooreÞeld, Edinburg, TX 78541. 3 U.S. Horticultural Research Laboratory, Subtropical Insects Re- Knowledge of the spatial distribution of insects is search Unit, USDAÐARS, 2001 South Rock Rd., Fort Pierce, FL 34945. important in understanding the biology and ecology of August 2008 SETAMOU´ ET AL.: SAMPLING PLAN FOR ASIAN CITRUS PSYLLID 1479 a species. In addition, information on the spatial dis- of the whole orchard. Subsequently, the canopy of the tribution of an insect pest is the basis for the devel- selected trees was divided into four quadrants along opment of a reliable sampling plan (Binns and Nyrop the four cardinal points. Five new ßushes were ran- 1992), which is required for the implementation of a domly sampled in a nondestructive manner for each of successful integrated pest management (IPM) pro- the southeast (SE), southwest (SW), northeast (NE) gram. Sampling is performed to quantify pest densi- and northwest (NW) canopy quadrants, for a total of ties, to deÞne treatment thresholds, and to forecast 20 ßushes per tree. First, ßushes were carefully ex- pest populations. Researchers also need reliable sam- amined and the number of D. citri adults per ßush was pling procedures to describe pest populations. counted and recorded. Using a 10ϫ hand held lens, Dispersion patterns of and sampling plans for D. citri nymphs and eggs also were counted in situ and re- have been investigated in the United States in plant- corded per ßush. No attempt was made to distinguish ings of orange jasmine, Murraya paniculata (L.) Jack between the Þve instars of the psyllid. (Tsai et al. 2000). No research information on sam- A ßush shoot was recorded as infested whenever at pling citrus ßush in the United States was available. least one of any developmental stage of D. citri was Dharajothi et al. (1989) studied sampling for D. citri in present. The percentage of infested ßushes was cal- citrus in India by using a sample unit of one new ßush culated per tree. The mean number (m)ofD. citri shoot (4Ð5 cm in length). Eggs and nymphs were eggs, nymphs or adults and the related variances (s2) found to follow an aggregated dispersion among sam- per ßush per tree were calculated for each orchard. pling units per tree, whereas adults were found to A nested analysis of variance (ANOVA) (PROC more often follow a random dispersion (Dharajothi et NESTED, SAS Institute 1999) (Zar 1999) was con- al. 1989). These researchers projected that 40, 38, or 19 ducted to determine the variance components of each sample units per tree for eggs, nymphs or adults would hierarchical level of the sampling plan (ßushes nested provide a sufÞcient level of precision for a mean den- within canopy quadrant nested in trees nested in or- sity prediction. Due to possible differences in citrus chard nested in host plant type) and their relative culture, climate, and other factors among citrus in- contribution to the total variance in psyllid infestation dustries around the world, the appropriateness of data and densities on ßush shoots. Because different num- presented by Dharajothi et al. (1989) relative to D. bers of orchards were sampled for each of the variety citri infestations in the United States is not known. and thus the design was unbalanced, PROC NESTED The aim of this research was to study the population provided only unbiased estimates of variance compo- densities and spatial distribution patterns of D. citri on nents. To determine the signiÞcant level of each citrus ßush and to determine the number of ßush nested factor, a mixed model analysis with the TYPE samples needed to make reliable estimates of its pop- 3 estimation of mean square was used (PROC MIXED, ulation densities on citrus. This information is re- SAS Institute 1999). Subsequently, the RyanÐEinotÐ quired for the development of management decisions GabrielÐWelsh (REGWQ) test was used to discrimi- of the pest in citrus orchards. nate between means of Þxed factors, i.e., host plant and quadrant (Westfall et al. 2003). All D. citri counts were log (x ϩ 1)-transformed, and percentage of ßush Materials and Methods infestation per tree was arcsine transformed before Data Collection. Thirty-four commercial grapefruit, analysis to correct for nonadditivity and non-normal- Citrus paradisi Macfad., and six sweet orange, C. si- ity, respectively. nensis (L.) Osbeck, citrus orchards were sampled for Within-Tree Dispersion Analysis. Spatial distribu- all development stages of D. citri from March to Au- tion patterns of the life stages of D. citri among ßushes gust 2006 in southern Texas. These orchards were per tree were determined using the IowaÕs patchiness located in the Texas citrus belt in the three southern regression (1968) and TaylorÕs power law (Taylor most counties (Hidalgo, Cameron, and Willacy coun- 1961) for each of the two citrus types, grapefruit of ties) of the Lower Rio Grande Valley of Texas, where sweet orange. TaylorÕs power law model expresses Ϸ11,000 ha of commercial citrus are grown in an area varianceÐmean relationships and provides a quan- extending Ϸ50 km from north to south and Ϸ 80 km titative analysis of the index of aggregation in terms from east to west. The sample of citrus orchards was of some ecologically meaningful parameters (Kuno assumed to be representative of the whole survey area. 1991). Each orchard sampled had a minimum acreage of 2 ha IowaÕs patchiness regression quantiÞes the relation- (range, of 2Ð24 ha and an average of 5.2 ha). Selected ship between the mean crowding index M and the orchards varied in age but each had profuse new ßush sample mean m as follows: growth at the time of sampling.