Repellent effect of guava leaf volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus

Insect Science () 17, 39 5, DOI.1111/j.17-7917.9.171.x Repellent effect of guava leaf volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus Syed Muhammad Zaka 1, Xin-Nian Zeng 1, Paul Holford and George Andrew Charles Beattie 1 Key Laboratory of Natural Pesticide and Chemical Biology of the Ministry of Education, South China Agricultural University, Guangzhou, China, Centre for Plant and Food Science, University of Western Sydney, Locked Bag 1797, Penrith South DC, New South Wales 1797, Australia Abstract The Asiatic citrus psyllid (Diaphorina citri Kuwayama [Hemiptera: Sternorrhyncha: Psyllidae] is a vector of huanglongbing (citrus greening), a devastating disease of citrus caused by phloem-limited bacteria. Growing guava (Psidium guajava) asaninter- crop appears to be a successful means of reducing psyllid numbers within citrus orchards; however, the mechanism by which such a reduction is achieved is unknown. To determine the repellent effect of guava leaf and factors attributed to this activity, responses of adult psyllids to guava leaf and its odor were evaluated in cage tests and Y-tube olfactometer test. The results showed that guava leaf possessed a repellent effect against the adult citrus psyllids. Fewer psyllids were found on citrus leaves in the presence of guava foliage than in its absence. Young and old guava leaf showed equal repellent activity. By covering the guava shoots with net cloth, it was revealed that the repellent effect of guava leaf against adult psyllids on citrus was attributed to the volatile compounds, rather than physical factors. The olfactometer response of adult psyllids to guava leaf odor was dosage-dependent. Between guava odor and control, only 35.%, 5.% and 1.5% of the psyllids moved toward guava odor when presented with 5.,. and 15. g of guava shoots, respectively. The olfactometer experiments also showed that both male and female psyllids responded similarly to the guava leaf odor. Key words Asiatic citrus psyllid, huanglongbing (HLB), insect behavior, Psidium guajava, volatile compounds Introduction The Asiatic citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psyllidae) is a major pest of citrus (Halbert & Manjunath, ; Yang et al., ). The psyllid prefers to feed on the phloem of tender shoots and leaves and can induce abnormal growth and shoot dieback (Husain & Nath, 197; Bové, ; Halbert & Manjunath, ; Michaud, ). The insect also ex- Correspondence: Xin-Nian Zeng, Key Laboratory of Natural Pesticide and Chemical Biology of the Ministry of Education, South China Agricultural University, Guangzhou 5, China. Tel: + 595; email: zengxn@scau.edu.cn cretes copious amounts of honeydew on which sooty mould may grow (Reynolds, 1999). However, the Asiatic citrus psyllid is a major pest mainly because it is the major vector of huanglongbing (HLB) or citrus greening, a devastating disease caused by phloem-limited, Gram-negative liberibacters (α-proteobacteria). In several Asian countries, in Papua New Guinea and the USA, the disease is caused by Candidatus Liberibacter asiaticus (Bové et al., 199; Garnier & Bové, 199; Weinert et al., ); in Africa by Ca. L. africanus; in Mauritius and Réunion by Ca. L. asiaticus and Ca. L. africanus (Garnier et al., 199); and in Brazil by Ca. L. asiaticus and Ca. L. americanus (Yamamoto et al., ). The disease leads to unmarketable fruit, reduced yield and fruit quality, tree dieback, and eventual death of infected trees (McClean & Schwarz, C 9 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences 39

S. M. Zaka et al. 197; da Graça, 1991; Bové, ). The host range of D. citri includes species and varieties of Citrus and Citrus relatives including Murraya paniculata (L.) Jack and M. exotica L. (both known as orange jasmine) of the tribe Aurantieae [Sapindales: Rutaceae: Aurantioideae], Bergera koenigii L. (curry leaf), and some species of Clausena including Cl. lansium (Lour.) Skeels (wampee or huangpi) of the tribe Clauseneae [Aurantioideae]. Using insecticides for the control of D. citri limits, but does not prevent, the spread of the disease, and biological control is not feasible given the serious nature of the disease and the effectiveness of disease transmission. In Vietnam it has been observed that citrus orchards planted with guava (Psidium guajava L. [Myrtales: Myrtaceae]) showed much lower psyllid infestation levels and low incidence of HLB compared to citrus orchards lacking guava (Beattie et al., ). Although raising guava as an intercrop appears to reduce psyllid numbers in orchards, the mechanism by which this occurs is unknown. The effects of guava on citrus psylla could be due to physical disruption on host recognition, repellent effect of volatile compounds from guava or chemical alteration of the volatile compounds emitted by citrus reacting with guava compounds. The present study was conducted to determine if guava has a repellent effect on adults of Asiatic citrus psyllids, the attributions (chemical or physical) of repellence, dose response of adults to guava volatiles, and whether the volatile compounds from guava alone, or by reacting with that of citrus, play the role. Materials and methods Insects Adults of D. citri were collected daily from -yearold sweet orange (Citrus aurantium L.) trees in the botanical garden of South China Agricultural University, Guangzhou, China. High psyllid populations were maintained by pruning the trees regularly to encourage new growth favored by the psyllid. Adults were collected with a mechanical aspirator each morning and held in small plastic cups for use later in the day. Males and females were separated on the basis of their morphology. The tip of the abdomen of an adult male is bent upwards while the abdomen of the female is straight. Plants Four-year-old guava (Psidium guajava L. cv. Pearl) and sweet orange trees from the university campus were used as the source of plant materials. These plants were regularly irrigated and fertilized and were visually free from any disease or pest. Fresh new shoots of both guava and citrus were collected at the same time from the field for experimental use. Repellent effect of guava shoot The responses of mixed populations of adult male and female psyllids to guava and citrus shoots (15. cm in length) with fresh leaves were observed in no-choice experiments in which adult psyllids were released into cages (. cm 3. cm. cm) containing the same treatment. In the first test, one guava shoot was placed aside a citrus shoot as a treatment in a 5-mL flask moistened inside with some tap water. Five flasks containing the guava and citrus shoots were placed in one cage and five containing citrus shoot only were placed in another cage as control. The flasks were positioned randomly, but equidistant from each other, within the cage. Fifty adults (5 males and 5 females) were released into each cage and kept at ambient laboratory conditions. The number of adults settled on citrus leaves was recorded after 1 h and h. Each assay was replicated five times. In the second test, the repellent effect of mature and immature guava shoots on the settlement of adults on citrus was measured by using guava shoots with leaves fully hardened and expanded, and leaves soft and not fully expanded. Also, in order to determine that the repellent effect of guava is attributed to the odor (volatiles) instead of physical factors (leaf color, shape and structure), the response of adults to covered and uncovered guava shoots was measured using the method mentioned above. Guava shoots were covered with fine white net cloth, allowing volatile compounds to go through and preventing psyllids from being affected by the color, shape or structure of guava leaves. Repellent effect of guava leaf volatiles To confirm the repellent effect of guava leaf volatiles on adult psyllids, Y-tube olfactometer responses of adults to guava leaf odor were measured based on the methods of Horton & Landolt (7). The arms were 1. cm long and.5 cm in diameter. Charcoal-filtered and distilled water-moistened air was vacuumed through the system at a rate of 5 ml/min. The choice combinations of odor sources of guava, guava plus citrus, citrus (control), moist air (blank) were tested. A line was drawn on each of the two arms at. cm distance from the arm junction. Psyllids crossing the line within min spells were C 9 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 39 5

Repellent effect of guava leaf volatiles 1 considered to have made a choice. Each combination was measured using psyllids individually and was repeated eight times by changing new materials and shifting arm positions alternately. All the experiments were done under laboratory conditions at temperatures of 3 C and lamp-light intensity of mw/cm. In the dosage response experiment, choices of mixed populations of male and female psyllids to volatiles from.5 g, 5. g,. g, and 15. g of guava leaves were compared to a blank. Also, the choices to volatiles from the same series amount of guava leaves plus 5. g of citrus leaves were compared to volatiles from 5. g of citrus leaves. Response of adult males and females to volatiles emanating from 15. g of guava leaves versus blank was also evaluated separately using the same methods. Statistical analysis Differences between treatments were determined using repeated-measures analysis of variance (ANOVA) with the time of assessment being the repeated measure using Statistica (Version 7, StatSoft, Inc., Tulsa, OK, USA). All data sets were homoscedastic according to Levene s test and means were separated using Tukey s Honestly Significant Difference (HSD) test. Contingency χ tests (including Yates correction) were used to determine whether the numbers of psyllids moving toward either of the two arms of the olfactometer were significantly different. Results Repellent effect of guava shoots The results showed that significantly fewer psyllids were found on citrus leaves in the presence of guava foliage than in its absence (F = ; P <.1) (Table 1). The percentage reductions of adult numbers were 3.% and 5.7%, respectively, after 1-h and -h treatments. It indicates that guava shoots possess repellent effects against the adults of citrus psyllids. There was a significant difference in the mean number of adult psyllids settled on the citrus leaves between the treatments having guava and no guava (Table ). However, no significant difference could be seen between the mean number of psyllids on citrus with immature and mature shoots, showing that they had equal repellent activity. By covering the guava shoots with net cloth, it was revealed that the repellent effect of guava shoots to adult psyllids on citrus was attributed to the volatile compounds, rather than physical factors. The covered Table 1 Effect of guava shoot on the number of adult psyllids settling on citrus shoot. Mean adult no. ± s.e. per shoot (n = 5) Treatment 1 h h Citrus shoot + 9. ±.71 a 7. ±.55 a guava shoot Citrus shoot 1. ±. b 1. ±.73 b Means in the same column followed by same letter are not significantly different according to Tukey s HSD test at P =.5. Table Effect of guava shoot maturity on the number of adult psyllids settling on citrus shoot. Mean adult no. ± s.e. per shoot (n = 5) Treatment 1 h h Citrus shoot + mature. ±. a. ±. a guava shoot Citrus shoot +. ±. a. ±. a immature guava shoot Citrus shoot. ±.37 b 7. ±. b Means in the same column followed by the same letter are not significantly different according to Tukey s HSD test at P =.5. Table 3 Effect of covered guava shoot on the number of adult psyllids settling on citrus shoot. Mean adult no. ± s.e. per shoot (n = 5) Treatment 1 h h Citrus shoot + covered. ±.37 a 3. ±. a guava shoot Citru shoot + 3. ±.37 a 3. ±. a uncovered guava shoot Citrus shoot 7. ±.5 b 7. ±.51 b Means in the same column followed by the same letter are not significantly different according to Tukey s HSD test at P =.5. and uncovered guava showed no significant difference in the mean number of psyllids present on the citrus leaves, although a highly significant difference (F =., P <.1) occurred between both guava treatment and the no-guava control (Table 3). C 9 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 39 5

S. M. Zaka et al. A B C D Guava leaves (.5 g) Guava leaves (5. g) Guava leaves (. g) Guava leaves (15. g) R1 R R3 R R5 R R7 R Mean Fig. 1 Response of adult psyllids to volatiles emanating from different quantities of guava foliage. Repellent effect of volatiles from guava leaf The olfactometer response of adult psyllids to guava leaf odor was dosage-dependent (Fig. 1). When guava leaf odor and moist air (blank) were provided, no significant difference between the mean number of psyllids in the two arms was found (P =.35, Fig. 1A) at.5 g treatment. It was suggested that low concentrations of guava have no repellent effect on adult psyllids. When the amount reached 5. g of guava leaves, a highly significant difference between the mean number of psyllids in the two arms (P <. 1, Fig. 1B) was observed. The percentages of psyllids moving toward guava odor were 35.%, 5.% and 1.5%, respectively, in treatments of 5.,. and 15. g of guava shoots. When guava plus citrus and citrus leaf odors were provided in the two arms, similar, but stronger repellent activities were obtained. All treatments showed significant differences between the mean numbers of psyllids in the two arms (Fig. ). The percentages of adults moving toward the arm having both citrus and guava odors were 3.75%, 33.75%, 17.5% and 13.75%, respectively. This might indicate that the repellent activity of volatiles from guava leaf would be increased by interacting with those from citrus leaf. Results shown in Fig. 3 reveal that the response of males and females to guava leaves (15. g) were similar. Discussion Citrus HLB has become a great threat to the world citrus industry (Bové, ; Zhao, ). So far, no successful cure for it has been reported. The key measures for limiting its epidemic spread are the use of disease-free seedlings and the suppression of psyllid populations to reduce pathogen transmission. Observations in Viet Nam suggested that citrus groves interplanted with guava trees showed low populations of citrus psylla and low incidence of HLB-infected trees (Beattie et al., ). Understanding the mechanism by which guava affects citrus psylla may lead to the proper use of guava in the control of HLB. The results obtained in the present study revealed that volatile compounds, instead of color and shape of the leaves, played a role in reducing citrus psylla populations on citrus, although it is possible that other factors such as the masking of citrus volatiles may contribute to the response of the psyllid. It indicates that certain volatile compounds in guava leaf could be developed as repellents for the management of citrus psylla. Guava fruits and leaves produce a wide range of volatile compounds, such as sesquiterpenes (Sagrero-Nieves et al., 199; Ogunwande et al., 3), aldehydes and alcohols (Idstein & Schreier, 195; Begum et al.,, ; Soares et al., 7). Some of these aldehydes and alcohols are the socalled green leaf volatiles that have been shown to have repellent effects on insects (Jang & Light, 1991). Further studies are required to determine the active compounds in C 9 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 39 5

Repellent effect of guava leaf volatiles 3 A B C D Citrus leaves (5. g) Citrus leaves (5. g) + Guava leaves (.5 g) Citrus leaves (5. g) Citrus leaves (5. g) + Guava leaves (5. g) Citrus leaves (5. g) Citrus leaves (5. g) + Guava leaves (. g) Citrus leaves (5. g) Citrus leaves (5. g) + Guava leaves (15. g) R1 R R3 R R5 R R7 R Mean Fig. Choice response of adult psyllids to volatiles emanating from citrus foliage alone and from citrus foliage plus different quantities of guava foliage. guava leaves repelling D. citri and their efficacy. Research is also required to determine how guava, other plants that may produce volatiles that repel citrus psyllids, or perhaps synthetic repellents, can best be used to reduce the impact of HLB in citrus orchards. A. Male adults Guava leaves (15. g) B. Female adults Guava leaves (15. g) R1 R R3 R R5 R R7 R Mean Fig. 3 Repellent effect of guava leaf volatiles on male and female adult psyllids. Pre-alighting discrimination of a potential host plant implies that the relevant characteristics are perceived at a distance. As such, these cues are expected to influence orientation and choice by the insect. To investigate the chemical basis of pre-alighting discrimination, olfactometers that deliver volatiles to test insects while they are airborne have been developed and used with success (Eigenbrode & Bernays, 1997). With psyllids, Horton & Landolt (7) used a Y-tube olfactometer to show that males of the pear psylla, Cacopsylla pyricola (Förster) [Psyllidae], are attracted to volatile odors from femaleinfested or previously infested shoots. Cen et al. using a four-arm olfactometer demonstrated that D. citri females were strongly repelled by odors from essential oil of guava leaf and freshly ground guava leaf, but were not repelled by odor of essential oil of guava fruit (Cen et al., pers. comm., 7). In the present study, the olfactometer results revealed that the repellent action of guava against citrus psylla is dose-dependent, with very low doses having little effect on citrus psylla. This result indicates that to control citrus HLB by interplanting guava trees in citrus groves, sufficient numbers of guava trees are needed to keep the dosage of volatile compounds emitted from guava at an effective level in the entire grove. In China, C 9 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 39 5

S. M. Zaka et al. observations revealed that even in the presence of guava trees scattered inside or around the groves, citrus psylla populations are high. This could be due to the fact that the guava trees present did not release enough active volatile compounds. In Vietnam, it was suggested that guava trees are interplanted prior to citrus at a ratio of one guava tree to one citrus tree (Beattie et al., ). Besides, guava interplanting may increase severity of fruitfly damage to citrus (Xie & Zhang, 5; Xu et al., 5). Under these circumstances, fruit-removing or bagging would be adequate before guava fruits ripen to block the food chain of fruitfly in groves and to protect citrus fruits from fruitfly damage. Guava is a widely planted tropical and subtropical fruit. Guava leaf is traditionally used as an antidiarrheal drug. The oral acute toxicology of leaf extract to mice is low with a LD 5 of more than. g/kg (Aimmanas et al., 1995). 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