Resistance of Citrus and Related Genera to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Neotrop Entomol DOI 10.1007/s13744-014-0230-0 PEST MANAGEMENT Resistance of Citrus and Related Genera to Diaphorina citri Kuwayama (Hemiptera: Liviidae) PC BORGONI 1,JDVENDRAMIM 1,ALLOURENCÃO 2,MAMACHADO 3 1 Depto de Entomologia e Acarologia, ESALQ/USP, Piracicaba, SP, Brasil 2 Instituto Agronômico (IAC), Campinas, SP, Brasil 3 Centro de Citricultura Sylvio Moreira, IAC, Campinas, SP, Brasil Keywords Asian citrus psyllid, host plant resistance, huanglongbing Correspondence AL Lourencão, Instituto Agronômico (IAC), 13012-970 Campinas, SP, Brasil; andre@iac.sp.gov.br Edited by Jorge B Torres UFRPE Received 24 January 2014 and accepted 23 June 2014 * Sociedade Entomológica do Brasil 2014 Abstract The present study was developed to evaluate the resistance of the following genotypes of Citrus and related genera to this pest: Pera, Natal, and Washington Navel oranges (Citrus sinensis), Marsh Seedless grapefruit (Citrus paradisi), hardy orange Rubidoux (Poncirus trifoliata), kumquat (Fortunella margarita Swingle), citrumelo Swingle (C. paradisi x P. trifoliata), and citrange Troyer (P. trifoliata x C. sinensis). The experiments were performed in greenhouses with plants grafted onto Rangpur lime (Citrus limonia) and placed individually in voile cages. The preference for oviposition in a no-choice test, and the effect of genotype were evaluated. The egg-adult cycle was monitored to determine the effect of genotype on the biology of the insect. Poncirus Rubidoux was the least preferred genotype for oviposition; reduced number of eggs was also found to occur on citrange Troyer, and Marsh Seedless was the genotype with the most eggs. No significant variation in the duration of the embryonic period was observed; however, a difference in the viability of eggs was found, with the lowest egg viabilities on Swingle. Kumquat and Marsh Seedless genotypes were correlated with increased durations of the nymphal phase, however, there was no difference in the survival of this phase. Fecundity of females on Troyer, Swingle, and kumquat was reduced. Considering all of the evaluated parameters, it was concluded that cultivars of sweet orange are the most susceptible genotypes to Diaphorina citri. Regarding oviposition, P. trifoliata Rubidoux showed resistance of the antixenosis type. Introduction The increasing production costs in the citrus industry are associated with the challenge to control diseases and pests, among them huanglongbing (HLB). HLB or citrus greening is a disease that has been correlated with the phloem-restricted bacteria Candidatus Liberibacter spp. (Étiennea et al 2001, McKenzie & Puterka 2004). Both Ca. Liberibacter asiaticus and Ca. L. americanus are associated with HLB in Brazil (Coletta-Filho et al 2005). This disease is the main cause of the reduction of citrus orchards in Africa and Asia (Da Graça 1991, Parra et al 2003) and can lead to a total loss of production (Martinez & Wallace 1967). The symptoms of huanglongbing are stunting of trees, sparse leaves, unseasonal bloom, and dropping of leaves and fruit. Young leaves may appear chlorotic, while mature leaves may have a blotchy-mottling appearance and may develop vein corking. Typical HLB symptoms in the fruit include reduced size, lopsidedness, uneven coloring, rind hardness, poor quality juice, and aborted seeds (Mead 2002). HLB was detected for the first time in Brazil in orchards in the state of São Paulo in July of 2004 (Coletta-Filho et al 2004). The bacteria associated with the disease are transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama,

Borgoni et al which is common in Brazilian orchards (Silva et al 1968, Yamamoto & Gravena 2000). The insect is also common and usually abundant on the exotic ornamental plant, orange jasmine (Murraya paniculata) (Sapindales: Rutaceae), which is considered one of its best hosts. In addition to being the HLB vector, D. citri is a sap-sucking insect that can cause damage such as leaf curling and twisting of shoots. If the infestation is intense, the stems dry up and the year s production may suffer substantial reduction. Michaud & Olsen (2004) reported that damage during the initial development of plants is related to the injection of toxins during nymph feeding, sometimes requiring the insecticide application to prevent plant damage. Research has shown genetic variability in resistance to D. citri in Citrus species and other Rutaceae plant species used by the psyllid as host plants (Chakravarthi et al 1998, Tsai & Liu 2000, Navaet al 2007, Ikeda & Ashihara 2008, Westbrook et al 2011, Richardson& Hall 2013), which illustrates the importance of the present study in evaluating resistance of the most important citrus genotypes and their hybrids to D. citri in the more in Brazil. Even though D. citri has been known to occur in Brazil since the 1940s (Costa Lima 1942), no studies have reported its preferences for various citrus genotypes. Therefore, the aim of this research was to evaluate the preference for oviposition in no-choice tests and the effects of host plant genotype on the biology of the insect. Material and Methods Biological traits of D. citri were evaluated on the following rutaceous genotypes: Citrus sinensis (sweet orange) cv Pera, Natal, and Washington Navel, Citrus paradisi (grapefruit) cv Marsh Seedless, Poncirus trifoliata (poncirus) cv Rubidoux, Fortunella margarita (kumquat), P. trifoliata x C. sinensis (citrange) cv Troyer,and C. paradisi x P. trifoliata (citrumelo) cv Swingle. Budwood of these genotypes was grafted onto Rangpur lime (C. limonia), and the trees were grown in 3-L plastic bags within a screen house. Diaphorina citri rearing To supply insects for bioassays, D. citri was reared on orange jasmine (M. paniculata) in a greenhouse. The reared insects were kept in 50 90 50 cm aluminum cages covered with an anti-aphid screen. Orange jasmine plants were cultivated in 0.5-L plastic bags and pruned after blooming. Fifty centimeters long stems were used for oviposition and feeding. Plants were kept in the oviposition cages for 2 to 3 weeks, after which they were replaced by new ones. Plants with eggs and nymphs were transferred to new cages (emergence cages for the adults), and emerged adults were collected and transferred to oviposition cages every 2 days. After plant removal, the cages were washed to reduce the incidence of entomopathogenic fungi. After emergence of all adults, the orange jasmine plants were removed from the cages, pruned, and sprayed with imidacloprid (Provado 200 SC ) for their future reuse in the oviposition cages. Oviposition in no-choice tests This experiment was carried out in screened (voile) cages (50 80 50 cm) with a 20-cm Ø opening to facilitate plant and insect manipulation. Two plants of each genotype were placed in each cage, and each plant had suitable flushes for oviposition. One cage per treatment (genotype) was used. Fourteen couples of D. citri were released in each cage. Each treatment was replicated in five blocks. The adults were kept for 72 h to allow oviposition, and the number of eggs per plant was counted. Egg to adult development The experiment was carried out in a screen house in voile cages with two bloomed plants for adult oviposition on the genotype to be evaluated. In order to obtain a sufficient number of eggs on both plants in the cages, 40 adult insects were released into each cage. After 72 h, the adults were removed, and the number of eggs hatched, dead nymphs, and emerged adults were counted daily for the determination of the duration and survivorship of the nymph stage. Emerged adults were sexed to determine the sex ratio and establish couples, and evaluated for deformities, especially in the wings. Fecundity Adult fecundity was evaluated under controlled conditions (27±1 C; 60±10% RH; 14 h photophase). Insects were maintained inside 2-L transparent polyethylene terephthalate (PET) cages carrying a voiled-sealed 30 cm 2 opening on the side for air exchange. The cage was placed on top of an 11-cm Ø Petri dish, holding a Rangpur lime seedling. Each seedling was infested by two newly-emerged couples of D. citri.each replicate was composed of two cages each containing a Rangpur lime plant and two couples from one of the tested genotypes, with a total of five replicates per treatment. Seedlings were replaced every 72 h and the mean number of eggs counted. The evaluations were performed until the female s death, and the female fecundity and female reproductive period were determined. In order to determine the duration and viability of the egg stage, eggs and live and dead nymphs were daily counted in each tube. For each treatment, 1,000 eggs were evaluated, distributed into five replicates of 200 eggs/each.

Citrus resistance to Diaphorina citri Statistical analysis Greenhouse experiments were performed in a randomized block design with five blocks, while those under BOD controlled conditions followed a completely randomized design, with five replicates/treatment. Data was subjected to ANOVA (p<0.05) and means compared by Duncan s multiple range test (p<0.05), with the exception of the fertility and embryonic development, which were compared using the Tukey s test(p<0.05). Results and Discussion Oviposition in no-choice tests In a no-choice test of oviposition, the least number of eggs were deposited on the Rubidoux genotype of P. trifoliata (Fig 1). Oviposition occurred in only one of the replicates involving this genotype, and, therefore, Rubidoux was excluded from the evaluation of the other parameters. Westbrook et al (2011) also found only a small number of eggs of D. citri on two P. trifoliata cultivars tested, and Richardson & Hall (2013) documented that most cultivars of P. trifoliata, including Rubidoux, are resistant to colonization by the psyllid. The number of eggs oviposited on Troyer citrange was significantly less than on either Washington Navel sweet orange or Marsh Seedless grapefruit. For the other genotypes, the number of eggs did no differ among them (Table 1). In studies carried out by Tsai & Liu (2000) with seedlings of four genotypes including orange jasmine and grapefruit, the highest preference for oviposition was on Marsh Seedless grapefruit. However, it is possible that grapefruit becomes less favored for oviposition as plants mature, as the population levels of D. citri were generally lower on mature grapefruit trees than on mature sweet orange trees (Hall et al 2010). And even though grapefruit is a common host, it is not preferred by D. citri for oviposition and feeding (Aubert 1987). Nevertheless, in the present study, kumquat was as preferred as Pera sweet orange. Also according to Aubert (1987), even though sweet orange is a common host of the pest, it is not as preferred as Murraya sp. or C. aurantifolia (Christm.) Swingle, the latter commonly known as Mexican lime or Key lime. Egg to adult development Despite the high avoidance of D. citri to oviposit in P. trifoliata cv. Rubidoux (Fig 1), the hatched nymphs normally developed, as they presented no deformities and survival did not differ from control ( Pera sweet orange) (Table 1). Nevertheless, Aubert (1987) asserted that P. trifoliata negatively affects nymphal development, thus some cultivars of P. trifoliata other than Rubidoux might exert some antibiosis to nymphs. Richardson & Hall (2013) reported that some cultivars of P. trifoliata exert antibiosis to adult psyllids. Development of nymphs on Marsh Seedless grapefruit and kumquat plants were similar to those on Swingle citrumelo (Table 1), and our data on eight genotypes were very similar to those reported by Liu & Tsai (2000) onfour genotypes. We did not detect any significant differences in survival of D. citri from nymph to adult among the eight tested genotypes (Table 1). The survival we reported was much lower Fig 1 Number of eggs per female of Diaphorina citri laid on young shoots of eight genotypes of citrus in no-choice tests.

Borgoni et al Table 1 Mean developmental duration and viability of the nymph stage of Diaphorina citri on eight citrus genotypes (27±1 C; 60±10% RH; 14:10 H L:D). Genotype Duration (days)±s.d. a Viability (%)±S.D. a Sex ratio Deformed adults (%) Kumquat 18.6±0.55 a 54.3±11.52 a 0.52 7.5 Marsh Seedless grapefruit 18.2±0.88 a 55.1±14.39 a 0.50 4.4 Swingle citrumelo 16.1±0.59 ab 65.0±4.69 a 0.50 5.3 Rubidoux P. trifoliata b 15.6 72.7 0.57 0.0 Washington Navel sweet orange 15.4±0.94 b 55.9±12.47 a 0.53 6.8 Pera sweet orange 15.1±0.67 b 73.7±12.02 a 0.50 3.1 Natal sweet orange 15.0±1.27 b 71.7±10.43 a 0.60 0.5 Troyer citrange 14.5±1.19 b 59.9±13.95 a 0.51 4.7 a Means in the same column followed by the same letter are not significantly different based on Duncan s multiple range test (p 0.5). b Genotype disregarded in the statistical analysis because of the low number of individuals. than those reported under controlled conditions (Liu & Tsai 2000), very likely due to the fact that the tests were developed using M. paniculata, which is considered a standard host for the development of D. citri. In evaluating data concerning the effect of citrus genotypes on the sex ratio of D. citri, wedisregardedthe Rubidoux genotype because only seven adults were obtained using this host plant. In six out of the seven remaining citrus genotypes, sex ratio of D. citri ranged from 0.50 to 0.53, but reached 0.60 on Natal sweet orange (Table 1). This suggests that some factor associated with Natal may influence male survival. There was no variation in the percentage of deformed adults (characterized by wing anomalies), with the highest values recorded on Washington Navel sweet orange (6.8%) and kumquat (7.5%) as compared to 3.1% for the control Pera sweet orange (Table 1). Fecundity Fecundity obtained on P. trifoliata cv. Rubidoux was excluded from analysis because it was not possible to obtain enough insects in 6 days. Among the other genotypes, female fecundity ranged from 15.5 eggs/female on Troyer citrange to 201.4 on Marsh Seedless grapefruit, as opposed to 106.5 eggs/female obtained on Pera sweet orange (control). Due to the data variability, there were no significant differences among the other genotypes with respect to fecundity (Table 2). Also, no significant differences were found in the mean period for oviposition between genotypes, which ranged from 13.2 to 17.2 days. Nava et al (2007) compared the fecundity of D. citri on Rangpur lime, Sunki mandarin, and M. paniculata, and they found that Sunki, with 166.2 eggs per female, was less preferred than M. paniculata (348.4 eggs). The numbers obtained for those species were very close to those we observed for Natal and Pera sweet oranges (Table 2). Aubert (1987) and Tsai & Liu (2000) found higher mean numbers of eggs per female than we observed, even for Marsh Seedless grapefruit, which is known as a good host of D. citri. This might be attributed to the fact that their insects were raised under field conditions as opposed to ours, which were lab reared. In our studies, many adults Table 2 Mean fecundity, mean duration of oviposition, egg viability and mean duration of development of the egg stage of Diaphorina citri on seven citrus genotypes (27±1 C; 60±10% RH; 14:10 H L:D). Genotype Number of eggs/female±s.d. a Oviposition period Viability (%)±S.D. a Duration (days)±s.d. a (days)±s.d. a Marsh Seedless grapefruit 201.4±19.88 a 14.7±1.16 a 79.4±3.06 ab 4.4±0.11 a Natal sweet orange 151.3±51.02 ab 16.7±3.12 a 78.4±2.73 ab 4.5±0.15 a Pera sweet orange 106.5±20.76 abc 13.2±1.50 a 87.4±2.13 a 4.2±0.13 a Washington Navel sweet orange 90.7±27.65 abc 15.0±2.05 a 75.0±3.60 ab 4.4±0.14 a Kumquat 70.5±16.10 bc 17.2±2.59 a 73.9±3.73 ab 4.5±0.10 a Swingle citrumelo 49.0±14.17 bc 14.4±2.08 a 67.7±7.56 b 4.4±0.20 a Troyer citrange b 15.5±12.50 c 15.9±8.44 a 75.0 4.3 a Means in the same column followed by the same letter are not significantly different according to Tukey s test(p 0.5). b Not included in the analysis of viability and duration of development of the egg.

Citrus resistance to Diaphorina citri introduced into cages died precociously, either not ovipositing or ovipositing a reduced number of eggs, which could have resulted in the reduced number of eggs per female as compared to those reported elsewhere (Liu & Tsai 2000, Tsai& Liu 2000, Mead 2002). So, probably this discrepancy was related to variations in temperature and humidity, and not to the host plant. For survival and duration of the egg stage, neither the data from P. trifoliata cv. Rubidoux nor from Troyer citrange were used because the number of eggs per female per plant were small, making the number of replicates insufficient for statistical analysis (Table 2). Egg viability on all seven genotypes was above 65%, and only the egg viability on Swingle citrumelo differed significantly from the control Pera sweet orange (Table 2). No significant differences were found among treatments in the duration of the embryonic period, which ranged from 4.19 to 4.50 days. The values for duration and viability at the egg stage we report are similar to those in the literature (Liu & Tsai 2000, Navaet al 2007). Considering all of the evaluated parameters, we concluded that cultivars of sweet orange are the most susceptible genotypes to D. citri, mainly cultivar Pera. Regarding oviposition, P. trifoliata Rubidoux showed resistance due to antixenosis. This fact hindered us from obtaining other biological data of D. citri in this cultivar. Thus, this citrus species can be considered as valuable germplasm for breeding programs aiming to develop D. citri resistant cultivars for the control of HLB. Acknowledgments This study was supported in part by a grant from CNPq and FAPESP. José Djair Vendramim, André Luiz Lourenção, and Marcos Antonio Machado are fellows of CNPq. 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