J Insect Behav (2015) 28:138–146 DOI 10.1007/s10905-015-9488-2

Host-Choice Behavior of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) Under Laboratory Conditions

Chuan-Qing Ruan & David G. Hall & Bo Liu & Yong-Ping Duan & Tao Li & Han-Qing Hu & Guo-Cheng Fan

Revised: 28 January 2015 /Accepted: 13 February 2015 / Published online: 25 February 2015 # Springer Science+Business Media New York 2015

Abstract The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the vector of the bacterial pathogens that cause Huanglongbing (HLB), considered to be the most devastating disease of citrus worldwide. Knowledge of the ACP’s host- finding behavior aids in our understanding of the epidemiology of HLB and in designing experiments to investigate host plant resistance to ACP. We present the results of

Highlights • Adult of Asian Citrus Psyllids (ACP) spent at least 9 hours to identify host (M. paniculata and C. reticulata)fromR. simsii, regarded as a non-host plant of ACP. • ACP adults were always observed on R. simsii, suggesting that adults regularly moved among the plant species. • Adult ACP showed a strong preference for settling on flush leaves as opposed to mature leaves or other plant locations • For ACP adults, larger flush shoots of host plant were as attractive as tiny new shoots. C.

D. G. Hall : Y.

T. Li Fujian Agriculture and Forestry University, Fuzhou, Fujian 350003, China e-mail: [email protected] J Insect Behav (2015) 28:138–146 139 research conducted to assess the ability of adult ACP to distinguish between a non-host plant [ simsii Planch. (: )] and two host plant species [Murraya paniculata (L.) Jack and BLugan^ Citrus reticulata Blanco (Sapindales: Rutaceae)] in a caged, free-choice situation. After being introduced into cages with the three plant species, more than 9 h elapsed before adult ACP made definite plant choices. Subsequently for more than 7 days, although adults were observed on R. simsii each time the plants were inspected, greater numbers were usually observed on M. paniculata or C. reticulata. Ultimately, most adults were on C. reticulata,interme- diate numbers were on M. paniculata, and few were on R. simsii. However, at least some ACP adults were always observed on R. simsii,suggestingthatadultsregularly moved within a cage among the plant species. Regular movement of adults between plants would favor dissemination not only of its population but also of HLB. Adult ACP showed a strong preference for settling on flush leaves as opposed to mature leaves or other plant locations, and larger flush shoots were as attractive as tiny new shoots. It is well known that host plant volatiles such as those associated with flush attract ACP adults and regulate how fast and where they settle on a plant.

Keywords Asian citrus psyllid (Diaphorina citri Kuwayama) . huanglongbing . host- choice behavior

Introduction

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the vectors of bacterial pathogens that cause Huanglongbing (HLB), the most devastating disease of citrus worldwide (Aubert 1990;daGraça1991). HLB systemically infects citrus trees and causes symptoms including yellowed shoots with blotchy mottled leaves, lopsided fruit sometimes with uneven ripening at the stylar end, and aborted seeds. HLB- infected citrus trees gradually decline in productivity and may die in 3 to 5 years depending on factors such as tree age and grove management practices (Bouffard 2006; Halbert and Manjunath 2004; Hall and Hentz 2011).Thepathogenassociatedwith HLB is a vector-borne, phloem-limited bacterium that to-date remains uncultured in vitro. Three distinct etiologic agents of HLB have been identified on the basis of the 16S rRNA gene sequence: Candidatus Liberibacter asiaticus (Las), Candidatus L. africanus (Laf) and Candidatus L. americanus (Lam) (Gotwald 2010). ACP is the vector of Las, both pathogen and vector are thought to have originated in Asia and that have spread to South, Central and North America. ACP also transmits Lam in Brazil as well as in Hunan, China (Gotwald 2010). The African psyllid Trioza erytreae (del Guercio) (Hemiptera: Triozidae) is the vector of Laf, which occurs in Africa (Gotwald 2010). ACP adults are responsible for spreading Las from infected to non-infected trees (Hall et al. 2013). Up to 100 % of ACP adults may acquire Las as they complete their immature developmental period on Las-infected host plants (Pelz-Stelinski et al. 2010). Even if ACP do not become infected with Las during their nymphal development, the adults may acquire the pathogen at a rate of up to 40 % following 5 weeks of feeding on infected plants (Pelz-Stelinski et al. 2010). Large groups of ACP adults can transmit Las to non-infected citrus trees at a high rate (Pelz-Stelinski et al. 2010). Chao et al. 140 J Insect Behav (2015) 28:138–146

(1979) reported that the spatial distribution of ACP had a high correlation with the spread of HLB. ACP adults may leap when disturbed and fly a short distance to other flush on the same or neighboring trees (Hall and Hentz 2011). An HLB epidemic spreads more frequently within trees and along rows in a grove than between rows, with long-distance spread occasionally occurring (Gotwald 2010;Shenetal.2013). Chiyaka et al. (2012) simulated ACP dynamics and epidemic development of HLB within a single tree. Suppression of ACP populations and use of HLB-free seedlings are key components of current integrated management of HLB (Yang et al. 2006; Grafton-Cardwell et al. 2013). However, ACP is prolific, short-lived, tolerant of temperature extremes, vagile and highly efficient in transmitting HLB path- ogens. These traits of ACP make it quite difficult to manage the pest and HLB using biological or chemical control (Grafton-Cardwell et al. 2013). Attention has been paid to screen Aurantioideae germplasms with specific resistance to ACP and the difference of susceptibility to ACP were found among citrus varieties (Westbrook et al. 2011). Knowledge of ACP behaviors to find suitable hosts for reproduction or feeding can benefit the practice of screening germplasms resistant to the psyllids. Previous studies revealed that ACP oviposits and develops solely on new flush shoots (Aubert 1990), and the adults preferentially feed on flush (Hall and Albrigo 2007). The odor and color of the new flush may play an important role in ACP activities such as detection, location and evaluation of potential host plants (Patt and Sétamou 2010; Wenninger et al. 2009). However, it is not clear what the reaction of ACP would be when they are exposed to plants that differ in susceptibility. More understanding of the reaction may help us to screen citrus genotypes with resistance to ACP. Therefore, we conducted experiments to observe the psyllid’s choice between: (1) two ACP host plant species [Murraya paniculata (L.) Jack or Citrus reticulata Blanco cv ‘Lugan’, (each species Sapindales: Rutaceae)] and one non-host species [Rhododendron simsii Planch. (Ericales: Ericaceae)], (2) mature leaves and young flush shoots, and (3) host plants varying with respect to abundance and age of flush.

Materials and Methods

Maintenance of Insects and Host Plants

A laboratory colony of ACP was initiated with approximately 100 adults collected during June, 2010 from a planting of M. paniculata at West Lake Garden in Fuzhou, Fujian, China (26°5′49″ N, 119°17′34″ E). The colony was maintained on potted M. paniculata held in meshed cages (120×60×50 cm) under controlled climate conditions: 27±2 °C, 60±7 % RH, 16 L:8D photoperiod with light density of approx- imately 3000 Lux. Adult ACP were allowed to oviposit and nymphs to develop. Upon emergence, new adult ACPs were transferred onto freshly potted M. paniculata seed- lings in cages (30×30×40 cm). One-year-old seedlings of M. panciculata and two- year-old (20–25 cm tall) C. reticulata and R. simsii seedlings were used in these experiments. J Insect Behav (2015) 28:138–146 141

Choice Between Host and Non-Host Seedlings

M. paniculata and C. reticulata are favored host plants of the ACP while R. simsii is regarded as a non-host. Six potted seedlings of each plant species were subjected to the experiment. The plants were growing in 2 L pots, were similar in height and canopy shape, and all had new flush shoots. Three seedlings, one from each species, were randomly placed in a regular triangle pattern in a meshed cage (60×60×120 cm). There were a total of 6 cages (replications) in the experiment. Each cage was held at 27±2 °C, 60±7 % RH, and a 16 L:8D photoperiod. Twenty ACP adults (no regard to gender) were aspirated into a 70 mL vial; three vials each containing 20 adults were placed ontotheflooratthecenterofeachcageandtheadultswereallowedto disperse. ACP were released into the cages at 14:00 h in the afternoon, and the number of adults on each plant was counted 3, 6, 9, 18, 42, 66, 90 and 186 h later.

Choice Among Plants Differing in Flush Age

Potted seedlings of M. paniculata differing in flush abundance and age were studied: (1) seedlings with tiny new flush buds <0.8 cm long, (2) seedlings with slightly older flush shoots (0.8 to 1.0 cm in length), and (3) seedlings with older flush shoots more than 1.0 cm in length. Three seedlings of each flush age category were placed in a regular triangle pattern in a meshed cage (60× 60×120 cm) under 27±2 °C, 60±7 % RH, and a 16 L:8D photoperiod. There were 6 cages (replications). Groups of 40 adult ACP (no regard to gender) were aspirated into 70 mL vials. Four vials each containing 40 ACP were placed onto the floor at the center of each cage and all adults (160 total) were allowed to disperse from the tubes to the plants. The number of adults on each seedling was counted every afternoon at 15:00 h for 6 successive days.

Proportion of ACP Adults Between Developed Leaves and Flush Shoots of Host Plants

Eighteen seedlings of M. paniculata similar in size and with new flush shoots were used in this experiment. The seedlings were placed in 6 meshed cages (60×60×120 cm) under 27±2 °C, 60±7 % RH, and a 16 L:8D photoperiod. Two70mLvialseachcontaining25ACPadults(noregardtogender)were placed on the floor at the center of the cage and the adults were allowed to disperse. There were 6 cages (replications). The number of adults on flush and mature leaves along each plant stem was counted every afternoon at 15:00 h for 6 consecutive days. The percentage of ACP adults between flush and mature leaves were counted for each host plant. The mean percent of ACP adults was further calculated for a host plant.

Statistical Analysis

Analysis of variance (ANOVA) and Duncan’s test were used to process the data. All statistics were carried out with DPS software (Tang and Feng 2002). 142 J Insect Behav (2015) 28:138–146

Results

Choice Between Host and Non-Host Seedlings

In this free-choice experiment during the first 9 h after ACP were released into cages, adult ACP initially dispersed to and settled not only on its two host plants (M. paniculata and C. reticulata) but also on R. simsii, regarded as a non-host plant of ACP. There were no significant differences among the three plants for the first 9 h with respect to the mean number of ACP per plant (Fig. 1). However, at 18 h after release, the mean number of adults per plant on R. simsii was significantly lower than that on citrus. At 42 h after release, there were significantly fewer adult ACP on R. simsii than on the other two plant species. At 90 and 186 h after the releases, significantly greater numbers of ACP were observed on C. reticulata than on M. paniculata.

Choice Among Plants Differing in Flush Age

Regardless of the age of flush on a plant, significantly fewer adult ACP settled on plants the first day after releases than on any subsequent observation date (Fig. 2). Mean numbers of adult ACP per plant did not differ significantly among plants with young, intermediate or older flush shoots on any observation date.

Choice Between Mature Leaves and Flush Shoots Within a Host Plant

A mean of 10.6 adult ACP was present on each plant 1 day after release (Fig. 3). On the second day after the release, the total number of adults per plant increased significantly and thereafter did not significantly change. As many adults were on flush shoots as on other plant parts on the first day after adults were released (Fig. 4). But on the second day, as total number of adults per plant increased dramatically, significantly fewer individuals were on flush shoots than on other sites within the same plant. After a significant increase on the third day, the percentage of adults per plant daily observed

40 Rhododendron Citrus Murraya c 35 b b c a 30 a ab b b 25 a b Diaphorina b a b 20 a a a 15 a a a a (adults per plant) 10 a a a 5 citri 0 Mean number of 3 6 9 18426690186 Hours after Diaphorina citri release Fig. 1 The number of Diaphorina citri adults settled on its host plants Murraya paniculata Band Citrus reticulata compared to numbers settled on the non-host plant Rhododendron simsii. The bars with similiar letters are not significantly different among the 3 tested plant species at the same time after ACP release at P<0.05 (one-facter ANOVA) J Insect Behav (2015) 28:138–146 143

90 Aa 80 70 Aa Aa 60 Aa Aa Aa Aa Aa Aa Aa Aa Aa 50 Aa AaAa Aa Aa S 40 Aa 30 Ba M Ba Ba 20 L

citri (adultscitri per plant) 10

Mean number of Diaphorina 0 1234567 Days after adults of Diaphorina citri release Fig. 2 Number of Diaphorina citri adults on Murraya paniculata seedlings differing in flush age: (S) seedlings with tiny flush buds; (M) seedlings with new flush shoots 0.8 to 1.0 cm in length; and (L) seedlings with flush shoots more than 1.0 cm in length). The bars with similar little letters are not significantly different among 3 flush ages within the same day after ACP release, and the bars with similar capital letters mean no significant difference among different days within same flush ages at P<0.05 (two-factors ANOVA) on flush varied unsignificantly in the range from 52.9 to 68.8 % over the flowing study days. From 3rd to 7th days after release, adults showed a significant preference for flush as compared to other plant locations.

Discussion

In a free-choice situation under our experimental conditions, more than 9 h elapsed before adult ACP made definite choices between R. simsii, M. paniculata and C. reticulata. Subsequently, although adults were observed on R. simsii each time the plants were inspected, greater numbers were usually observed on M. paniculata or C. reticulata. Ultimately after 90 to 186 h, most adults were on C. reticulata,interme- diate numbers were on M. paniculata, and few were on R. simsii.Halletal.(2008) reported that, in cage studies, adult ACP were slower to find and settle on citrus

60 ACP on flush 50 a a a ACP on other sites 40 a a a

30

20 b 10 Number of adults at the

indicated site (adults/plant) indicated site 0 1234567 Days after adults released Fig. 3 Total number of adults per plant, bars with the same letter are not significantly different at P<0.05 (one-factor ANOVA) 144 J Insect Behav (2015) 28:138–146

80 a a 70 a a a a 60 a 50

40 a a b b 30 b b the indicated site Percent at adults b 20 ACP on flush ACP on other plant sites 10 1234567 Day after adults released Fig. 4 Percentage of adults on flush shoots compared to other plant locations. For each day, data points with the same letter are not significantly different at P<0.05 (one-factor ANOVA)

(C. paradisi Macf.) when other plant species were present. These authors speculated that there may be plant volatiles as well as visual cues associated with an ACP host plant that attract psyllids from long or short distances, and such attractants might be masked or missing in cage studies, inhibiting initial ACP recognition of it host plants. The importance of plant volatiles in host plant colonization by ACP adults has been noted. Wenninger et al. (2009) reported that ACP adults exhibited olfactory responses to host plants in a Y-tube olfactometer. Patt and Sétamou (2010)reportedpositive responses of ACP adults to volatiles emitted by flush associated with M. paniculata and Citrus limon (L.) Burn. f. cv ‘Eureka’. Although the attractiveness of host plant essential oils to ACP adults remains undetermined, the repellent effect of non-host plant essential oils on the psyllid’s behavior has been reported. Volatile oils from the plant species Mikania micrantha H.B.K. (Asterales: Asteraceae), Lantana camera L. (Lamiales: Verbenaceae), Eupatorium catarium Veldkamp (Asterales: Asteraceae) and Wedelia chinensis (Osbeck) Merr. (Asterales: Asteraceae) showed significant repellen- cy to ACP adults, while the volatile oil of Eucalyptus citriodora Hook (Myrtales: Myrtaceae) showed neither an attractant nor repellent effect (Cen et al. 2005). The volatile from another non-host plant, guava (Psidium guajava L., Myrtales: Myrtaceae), possessed a repellent effect against the ACP adults (Zaka et al. 2010). On citrus leaves treated with essential oils from guava leaves, ACP adults spent a longer time on non- probing and saliva secretion and changed to feeding mainly on the xylem (Zhu et al. 2010). It may be the combination of the attractiveness of host plant odors and the repellent effect of non-host plant odors that allows ACP adults to differentiate R. simsii from M. paniculata and C. reticulata sooner than between the two host plant species. When evaluating plants for ACP resistance it may be best to include non-host plant species as controls, especially when the number of adults settling on a plant is part of the resistance criteria. In our study, the fact that at least some ACP adults were always observed on R. simsii suggested that adults regularly moved within a cage among the plant species –it was unlikely that any of these adults continually stayed on R. simsii, as adults have been shown to die within 6 days or less when confined without one of its host plants (Hall et al. 2008). Regular movement of adults from one plant to another could be stimulated by factors associated with an individual host plant (for example, presence or J Insect Behav (2015) 28:138–146 145 absence of flush, or competition with conspecifics or other species), but there could be an innate drive in ACP adults to regularly move between host plants (which would favor disease dissemination). In fact, routine adult ACP movement between host plants under field conditions has been noted (Hall and Hentz 2011). Adult ACP showed a strong preference for settling on flush leaves as opposed to mature leaves or other plant locations, and the age of the flush generally did not matter. Females oviposit strictly on very young flush leaves, and even moderately large flush shoots of M. paniculata can have terminal leaflets appropriate for oviposition. Working with Citrus depressa Hayata and M. exotica L., Yasuda et al. (2005) found that ACP adults colonized leaves of all ages but females preferred flush. The results obtained in the present study revealed that ACP adults were able to identify host plants from the non-hosts in a short period of time (9 h). The adults spend significantly more time to differentiate a host plant from another host plant than from non-host. This indicates that the time ACP spend to make choice between plants may be used as a criteria to estimate the relative resistance of different citrus varieties to ACP. Though ACP adults showed strong prefer- ence for settling on flush leaves compared to the mature ones, the present study showed that the total number of settling adults per plant were not significantly different among M. paniculata plants differing in flush ages. This implies that a little difference of flushing status may not obviously affect ACP settling behavior when plants at different flush status are placed at very short distance from each other. This implication is helpful because, in the practice of screening citrus germplasms with resistance to ACP, it is difficult to eliminate the difference of flushing status among different plant genotypes.

Acknowledgments The authors thank the financial support of the project BEvaluation of citrus germplasm for psyllid resistance in China^ (No: 6618-22000-037-01S) of US Department of Agriculture. This study was also partially funded by Provincial Natural Science Foundation of Fujian, China (2012 J01106) and Chinese Special Fund for Agro-scientific Research in the Public Interest (No. 201003067-05).

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