Sources of Zanthoxylum and Citrus

Six Z. clava-herculis plants were purchased in 1-gal (3.8 L) pots at a nursery in Davenport, Florida, 6 Z. coriaceum were obtained in 3-gal (11.4 L) pots from another nursery in Palmetto Bay, Florida, and 10 Z. flavum were purchased in 3-gal (11.4 L) pots from Homestead, Florida. Three M. paniculata in 3-gal (11.4 L) pots were obtained from the Biological Control Laboratory at the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (DPI). Zanthoxylum fagara were grown from cuttings made from 3 plants obtained from a nursery in Gainesville, Florida. ‘Duncan’ grapefruit (C. x paradisi), ‘Madam Vinous’ sweet orange (C. sinensis) and ‘Ponkan’ mandarin (C. reticulata Blanco) were grown from seeds in the greenhouse at the DPI. Seedlings and cuttings of these plants were placed in 4.0- by 19.5-cm plastic forestry tubes (“Conetainers” [Steuwe and Sons, Corvallis, Oregon, USA]) and inserted into a Conetainer rack. The Conetainers were filled with 165 mL (133 g) of commercial readymade growing medium (“®Metro-Mix 300 series”), SUN GRO Horticultural Distribution Inc., Washington, USA). The growing medium contained 30–40% horticultural grade vermiculite, plus bark, Canadian sphagnum peat moss, horticultural grade perlite, bark ash, dolomitic limestone and a wetting agent. Approximately 2 to 3 seedlings germinated from single seeds planted in each conetainer. Seedlings were thinned to one per pot after establishment. Seedlings were fertilized with Osmocote® 4 weeks after germination and thereafter as needed.

Colonies of Leuronota fagarae and Diaphorina citri

Leuronota fagarae and D. citri were maintained at DPI, Gainesville, Florida, and University of Florida Southwest Florida Research and Education Center (UF-SWFREC), Immokalee, Florida. Colonies of L. fagarae were initiated from collections made from Z. fagara plants in Collier County, Florida. Adults and nymphs were caged on tall Z. fagara plants grown in 5-gal (19-L) pots using sleeve cages made from fine mesh organdy. Colonies of D. citri were maintained on 1–2 feet (30–60 cm) tall potted M. paniculata plants covered with screened cages. Colonies of both psyllids were kept in separate glass houses maintained at around 27 °C and 60% RH.

Evaluation of Citrus and Zanthoxylum as Hosts for L. fagarae and D. citri

Separate no-choice experiments with L. fagarae and D. citri were conducted at DPI to evaluate response of single male female pairs to different host plants. Both psyllids were evaluated on 3 citrus species C. sinensis (‘Madam Vinus’), C. x. paradisi (‘Duncan’ grapefruit) and C. reticulata ‘(Ponkan’ mandarin) and 4 Zanthoxylum species Z. fagara, Z. flavum, Z. coriaceum and Z. clavaherculis. Leuronota fagarae also was evaluated against M. paniculata. Leuronota fagarae came from a colony on Z. fagara and D. citri from a colony on M. paniculata, and were 1-week old when experiments were initiated. Both experiments were designed as randomized complete blocks (RCB) with 4 replications and conducted at 24 °C.

A single pair of male and female L. fagarae was placed in each Conetainer planted with C. sinensis, C. x. paradisi, C. reticulata or Z. fagara and covered with a transparent cylindrical tube. Tubes were then covered with pieces of stretchy nylon mesh secured with a rubber band to prevent the insects from escaping. Zanthoxylum clava-herculis, Z. coriaceum, Z. flavum and M. paniculata were larger potted plants so psyllids were confined to single shoots in cages made from plastic cups and fine mesh organdy.

All shoots had young emerging leaves suitable for psyllid development and reproduction. The same procedure was used for experiment with D. citri. Insects were observed daily for survival and progeny. Dead psyllids were removed using a soft camel's hair brush and gender determined by observing the genitalia under a stereomicroscope. Newly emerged F1 progeny adults were identified on the basis of wing color which is much lighter compared with older adults.

Development and Reproduction of D. citri on Z. fagara

Three experiments were conducted at UFSWFREC to compare survival, development and reproduction of D. citri on the experimental host Z. fagara with ‘Swingle’ citrumelo and M. paniculata as controls. Four plants each of Z. fagara and ‘Swingle’ citrumelo were used in the first and second experiments. Zanthoxylum fagara plants were 4 feet (120 cm) tall in 5-gal (19-L) pots placed on the ground in a glasshouse maintained at around 27 °C and 60% RH. ‘Swingle’ citrumelo plants were 1.5-two feet (60 cm) tall in 1-gal (3.8-L) pots and placed on tables raised to the same height as Z. fagara. In the first experiment, 2 groups of adults that emerged on M. paniculata were evaluated for survivorship and reproduction on experimental plants. One group was allowed to feed upon emergence on M. paniculata for 3 d and the other for 1 week to increase the likelihood eggs would mature. Two shoots with emerging, unexpanded leaves suitable for development and reproduction of D. citri were confined individually on each plant using sleeve cages made from fine mesh organdy. Cohorts of D. citri adults containing an average 5 males and 4 females aged 3 days or 1 week were released in either cage and observed daily or every other day for survival and progeny. Dead psyllids were removed at each observation using the soft camel's hair brush and gender determined under a stereomicroscope. Once fifth instar nymphs were observed in any cage, parent psyllids were moved to another suitable shoot on the same plant and confined using a similar sleeve cage. Their progeny remained caged and F1 adults were removed and counted as they emerged.

For the second experiment, first, third and fifth instar nymphs of D. citri obtained from the colony on M. paniculata were evaluated for survival to adulthood on Z. fagara and ‘Swingle’citrumelo. Three shoots with newly opening leaves were selected on each of the 4 plants of each host, 1 each for first, third and fifth instar nymphs. Fifteen nymphs of the respective instar were released on each shoot and observed until they started crawling. Nymphs were removed gently from M. paniculata plants and placed on the experimental shoots using the soft camel's hair brush. Emerging adults were counted.

For the third experiment, adults obtained from nymphs reared on ‘Swingle’ citrumelo were evaluated for reproduction and progeny survival on Z. fagara and M. paniculata. Adults following emergence from each of the 3 age classes of nymphs on ‘Swingle’ citrumelo were divided into 6 cohorts resulting in total of 18 cohorts averaging 4–8 adults per cohort. These adults were placed in the freezer for 3–4 min to calm them for gender identification using a stereomicroscope and to make sure that sex ratio averaged 1:1. Cohorts then were caged individually on 18 young shoots of potted M. paniculata plants for 2 weeks to confirm that females were ovipositing before evaluation on Z. fagara against M. paniculata. The 18 cohorts were then distributed among 6 plants: 3 each of Z. fagara and M. paniculata with 3 cohorts caged per plant on 3 separate branches. Two weeks later, cohorts from Z. fagara were moved to M. paniculata to see if they were still able to produce viable progeny. Once fifth instar nymphs were observed in any cage, parent psyllids were moved to another suitable shoot and caged using a similar sleeve. Their progeny remained caged and F1 adults were removed and counted as they emerged.

Statistical Analysis

Data fitting a normal distribution as indicated by the Univariate procedure were analyzed by analysis of variance (ANOVA) General Linear Model Procedure (GLM) at a probability level of 0.05 (SAS Institute 2009, version 9.2). Mean separation for experiments with a large set of treatments was determined using Tukey's Studentized Range (HSD) test (D. citri or L. fagarae on 7 to 8 hosts) and for fewer comparisons using Least Significant Difference (LSD) test (D. citri only on ‘Swingle’ citrumelo, M. paniculata and Z. fagara) contingent on a significant F value for treatment effect. Non-normal data was analyzed using the non-parametric Kruskal—Wallis (SAS Institute, 2009, version 9.2).

Figures 1–5 are displayed in color in supplementary material for this article online in Florida Entomologist 97(4) (December 2014) at  http://purl.fcla.edu/fcla/entomologist/browse.

Biology of L. fagarae

Adults. The adult wild lime psyllid (Fig. 1A) is a small, brown-winged insect whose body is about 3.5 mm in length, including the wings. It has a pointed face, protruding downward, bright red eyes, and short antennae. Forewings are narrow and are marked with an irregular pattern of clear to yellowish-brown patches or spots of different colors. Typically, adults can live up to 1 ½ months and can be found aggregating on new flush where they feed and mate (Fig. 2). Gravid females have a patch of orange on a portion of the abdomen when eggs are ready to be laid (Fig. 3). Each female is capable of laying several hundred eggs, depending on the host plant. The average lifespan of an adult male and female wild lime psyllid is 46 days at 20 °C.

Wild lime psyllids sit at a 45 degree angle, similar to Diaphorina citri Kuwayama (Fig. 1B). The camouflage is excellent. Adult psyllids look like thorns on the stems and leaves of wild lime. Females spend most of their time on the young flush. We observed that if a female is active on a flush tip, a male will feed on older tissue 5–10 cm away. If the colony is crowded, more pairs will occupy the prime feeding and egg-laying locations.

Fig. 2.

Mating pair of wild lime psyllid Leuronota fagarae on the underside of Zanthoxylum fagara leaf.

Eggs. Adult females lay tiny almond-shaped eggs, which can be yellow-orange in color (Fig. 3). Wild lime psyllids lay their eggs on the leaf margins or on both sides of the stems (Fig. 3). The anterior and posterior ends of the eggs are attached firmly to the plant. The eggs can be arranged in a line where they can either be in single, double or triple rows. They become light brown just before hatching. At 20 °C, the average egg stage takes about 7 days.

Nymphs. The nymphs are wingless, flattened, and yellow with a transparent head, thorax and tip of abdomen. They are slow crawlers with bright red eyes and produce white waxy secretion (Fig. 4). The nymphal stage spans approximately 10-12 days. The nymphs molt 4 times before the final molt into an adult.

Fig. 3.

Female and eggs of wild lime psyllid Leuronota fagarae.

Fig. 4.

Nymphs of wild lime psyllid Leuronota fagarae and white waxy secretions they produce.

Damage caused by wild lime psyllids is similar to damage caused by other psyllids, especially some other triozids such as Leuronota maritima (Tuthill) on Avicennia and Trioza diospyri (Ashmead) on Diospyros. Feeding results in curling and twisting of the leaves, especially rolling of the leaf margins (Fig. 5) and shoot growth is reduced.

Longevity and Reproduction of L. fagarae

Longevity of males differed significantly among hosts (F = 45.32, df = 7, 24, P < 0.0001, Table 1). Average adult lifespan on Z. coriaceum was 46.5 ± 0.5 days and on Z. clava-herculis or M. paniculata was 41.5 ± 3.2 days which was significantly longer compared to all other hosts including Z. fagara on which this psyllid was first observed in Florida. Males lived an average of 16.8 ± 6.3 days on Z. fagara, significantly longer than 2.3 ± 0.5 days on Z. flavum. Male longevity on Z. fagara or Z. flavum did not differ statistically from any of the 3 citrus species which did not differ from each other (Table 1).

Fig. 5.

Young leaf of Zanthoxylum fagara curled by wild lime psyllid Leuronota fagarae.

Females lived an average 46.5 ± 0.5 days on Z. coriaceum which was similar to 41.5 ± 3.2 days on Z. clava-herculis and M. paniculata but significantly longer than on other hosts, including 35.00 ± 0.0 days on Z. fagara (F = 107.51, df = 7, 24, P < 0.0001, Table 1). Longevity was statistically similar among Z. clava-herculis, M. paniculata and Z. fagara but greater than Z. flavum or any of the 3 citrus species (Table 1).

Females lived as long or longer than males. Female longevity was double that of males on C. sinensis, C. x. paradisi and Z. fagara. F1 adults were recovered only on Z. fagara and averaged 26.3 ± 6.3 days per female.

Longevity and Reproduction of D. citri
Experiments Using Male Female Single Pairs

Male longevity was significantly affected by the host plant (F = 5.06, df = 6, 21, P = 0.0034, Table 1). Males lived an average of 29 ± 0.0 days on Z. clava-herculis significantly longer compared with all other hosts except 16.5 ± 7.3 days on C. sinensis and 10.5 ± 1.8 days on Z. coriaceum. There was no difference between these later two and the remaining hosts. The shortest lived males with an average life of 2.8 ± 1.8 days were observed on C. reticulata (Table 1). For some reason, our mandarin plants were not attractive for colonization, possibly due to the growth stage used.

Female longevity was also significantly affected by host plant (F = 6.45, df = 6, 21, P = 0.0009, Table 1). Longest lived females were observed on Z. clava-herculis with an average of 29 d, statistically similar to 19.3–20 days on Z. flavum, C. sinensis or C. x. paradisi but significantly longer compared with an average of 4-8 days on Z. fagara, Z. coriaceum and C. reticulata. The shortest lived females, averaging 3.8 ± 1.3 days of life, were observed on Z. fagara. F1 progeny matured on only C. sinensis and C. x. paradisi, resulting in 4.5 ± 0.5 and 3.5 ± 0.5 adults per female, respectively.

Effects of host plant on longevity of the longest lived individual were significant (F = 5.23, df = 6, 21, P = 0.0028). Generally, females lived the same or longer than males except on Z. fagara and Z. coriaceum (Table 1). Therefore, means and differences among means for longest lived individual were same as for females (Table 1).

Experiments Using Cohorts

Adult D. citri Longevity. Adults transferred to either Z. fagara or Swingle citrumelo lived an average of 68.7 ± 3.4 days and 64.2 ± 3.5 days following 3 days or one week on M. paniculata, respectively with no significant effect of time on the first host (F = 0.83, df = 1, 6, P = 0.3695). However, final host plant effects were significant with longevity averaging 76.2 ± 1.5 d and 56.7 ± 3.1 days on ‘Swingle’ citrumelo and Z. fagara, respectively (F = 35.53, df = 1, 6, P < 0.0001). Males and females on ‘Swingle’ citrumelo lived significantly longer than their counterparts on Z. fagara (Male: F = 36.09, df = 1, 6, P < 0.0001; Female: F = 10.76, df = 1, 6, P = 0.0073, Fig. 6) with a difference of 23 days and 16 days, respectively. All males survived through 13 days on ‘Swingle’ citrumelo compared to 8 days on Z. fagara, decreasing to 76% and 31%, respectively at 82 days (Fig. 7A). Female survival was 100% through 38 days on ‘Swingle’ citrumelo but only 10 days on Z. fagara and 82% and 55%, respectively, at 82 days (Fig. 7B).

Table 1.

Mean (± sem) longevity (days) of male (♂) and female (♀) Leuronota fagarae and Diaphorina citri adults on different plant species when tested by single male and female pairs. Leuronota fagarae used in the tests were reared on Zanthoxylum fagara and Diaphorina citri on Murraya paniculata and were one week old when experiments were initiated.

No F1 D. citri adults were recovered from cohorts on Z. fagara compared with 57 ± 14 per female on ‘Swingle’ citrumelo during 4–5 weeks after the move from M. paniculata (χ² = 12.89, df = 1, P = 0.0003). Cohorts with initial exposure of 3 days or 1 week on M. paniculata produced an average of 31.8 ± 5.5 and 82.3 ± 21.4 adults per female, respectively, on ‘Swingle’ citrumelo, without significant effect of the time of initial exposure (F = 4.02, df = 1, 6, P = 0.1388).

Nymphal D. citri Survival. Nymphs of D. citri released on Z. fagara did not develop to adulthood. However, emergence of adults from first, third and fifth instar nymphs released on ‘Swingle’ citrumelo averaged 61.3 ± 6.4%, 68.3 ± 7.4% and 83.3 ± 4.3%, respectively.

Effects of Host-Nonhost Transfers. Cohorts of D. citri adults reproducing actively on M. paniculata produced an average of 1.04 eggs per female during 2 weeks after being transferred to Z. fagara compared with parallel cohorts on M. paniculata which produced 19.02 eggs per female (Table 2). Nymphs hatching on Z. fagara during this period did not develop into adults, compared with 24.9 ± 8.9 per female that did so over the same interval on M. paniculata. When cohorts on Z. fagara were moved to M. paniculata, reproduction was similar to individuals left on M. paniculata, 51–55 adults per female (Table 2).

Implications for transmission of Las

Studies on citrus have shown that D. citri acquire Las more efficiently as nymphs than adults (Capoor et al. 1974; Inoue et al. 2009; Pelz-Stelinski et al. 2010). Acquisition times of 30 min for D. citri (Roistacher 1991) and 24 h for T. erytreae have been reported (Buitendag & von Broembsen 1993). Xu et al. (1988) found that acquisition by adults feeding of 5–7 h was sufficient to transmit citrus greening pathogens, while feeding periods of 1–3 h were not. Inoue et al. (2009) reported that Las apparently multiplies in D. citri when acquired by nymphs but not when acquired by adults. They also suggested that multiplication of the bacterium within the psyllids is essential for efficient transmission, and that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs. Pelz-Stelinski et al. (2010) found that adult D. citri that acquired the HLB pathogen as adults were poor vectors of the pathogen compared with adults that acquired the pathogen as nymphs. Considering high HLB infection rates in Florida citrus, most psyllid adults that end up utilizing Zanthoxylum as host could originate from nymphs that develop on infected trees.

Murraya paniculata is among the hosts that supported L. fagarae for the longest time (Table 1). However HLB symptoms or Las detection by Polymerase Chain Reaction (PCR) in M. paniculata is very rare in spite of its being a preferred host of D. citri. A host may be preferred for the vector but may not be preferred for the pathogens. For example, symptoms or PCR identification of HLB in M. paniculata, a preferred host to D. citri, are not common even in regions with high incidence of HLB (Tirtawidjaja 1981; Toorawa 1998; Hung et al. 2000; Deng et al. 2007). However, Laspositive D. citri nymphs have been documented on M. paniculata (Manjunath et al. 2008). Thus, even a non-preferred host of Las might be a source of positive psyllids.

Since acquisition of the HLB pathogen is largely the providence of nymphs, it appears to be unlikely that Z. fagara would ever serve as a source of Las acquired by D. citri. Pathogens transmitted to Zanthoxylum species by D. citri coming from infected citrus would be stuck in a dead end host unless L. fagarae was able to transmit Las among Z. fagara plants. Whether Zanthoxylum spp. plant would suffer from the disease is yet to be determined. In any case it seems unlikely that either L. fagarae or any Zanthoxylum species would present much of a threat for HLB spread to citrus, especially considering the relative rarity of these plants.

Diaphorina citri female adults survived on Z. flavum for 19 days, and on Z. coriaceum for 6 days, which is long enough for transmission of Las to these endangered plants. Their susceptibility to HLB is under investigation. If Z. flavum or Z. coriaceum did become infected, they are likely to be dead end hosts, because neither D. citri nor L. fagarae reproduced on them, and no other Rutaceae-feeding psyllids are known in Florida. Zanthoxylum flavum supported L. fagarae for the least amount of time compared with all other species and may be at less risk. Diaphorina citri females lived on Z. fagara up to 60 days, which is long enough for occasional transmission of Las. It remains to be seen if L. fagarae can acquire or transmit Las. The susceptibility of Zanthoxylum spp. to HLB pathogens and ability of L. fagarae to acquire and transmit these pathogens is under investigation.