Wood Apple, Limonia acidissima: A New Host For the Huanglongbing (Greening)Vector, Diaphorina citri Meisaku Koizumi, Maitree Prommintara, and Yoshihiro Ohtsu ABSTRACT. A study was conducted in Thailand to determine the host range of psyllid, Dia- phorina citri, and the huanglongbing (HLB) (greening) pathogen it transmits. Approximately six- month-old seedlings of 15 Rutaceae plants including three citrus cultivars were exposed to D. citri that had fed on HLB-infected citrus plants collected from Thailand. Long-term survival of the psylla of more than 7 wk was observed on the following plants: Balsamocitrus dawei, Murraya paniculata, M. koenigii, Limonia acidissima (wood apple), Atalantia sp., Severinia buxifolia, Pon- cirus trifoliata and Som-pan and Som-keo-wan mandarins. Among them, marked multiplication of psylla was noted on M. paniculata, Atalantia sp. and L. acidissima. The former two did not develop any symptoms, but the L. acidissima developed leaf mottling and yellowing. An electron microscope study failed to show conclusive evidence of HLB organisms in sieve cells of infected L. acidissima. These results indicate that wood apple is a new host for D. citri and warrants further investigation as a possible host of the HLB agent. Index words. Citrus huanglongbing disease, host range, Limonia acidissima, vector. Citrus Huanglongbing (HLB) tural Science (JIRCAS) and the (greening) disease is a major factor Thailand Department of Agriculture limiting citrus production in tropical (DOA). We found the build up of and subtropical Asia. An integrated large population of D. citri on the management program which wood apple, Limonia acidissima L. includes the propagation of disease- (= Feronia limonia). free plants, eradication of infected plants, and the effective control of MATERIALS AND METHODS the vector is recommended (1, 2). Identification of host plants for HLB Inoculum source. The following and its vector is important to four potted HLB-infected plants achieve this goal. were used: Special Mandarin from Until now, the pathogen has only Ratchaburi district, rough lemon been shown to infect certain Ruta- and Avon Ever Bearing (Calamon- ceae, namely Citrus species, Ponci- din) from Nan Horticultural Experi- rus trifoliata, and its hybrids. Other ment Station, and Madam Vinous genera among the Rutaceae have not sweet orange inoculated with a pum- been confirmed as hosts (5). On the melo source from ChaiNat. These other hand, the psyllid vector of plants showed typical HLB symp- HLB, Diaphorina citri Kuwayama, toms, namely, leaf mottling in all naturally lives on citrus cultivars plants and severe yellowing, stunt- and Murraya paniculata, although ing, and vein corking of Madam some other plants support its multi- Vinous. HLB organisms were con- plication (1). These experiments firmed in Special Mandarin and employed only a few rutaceous spe- Madam Vinous sweet orange by elec- cies. In addition, possible presence of tron microscopy. They were kept in ecotypes or races of D. citri is sug- an incubator at 26-28°C (night and gested (1). This study was carried day) with artificial illumination at out in Thailand to determine the 10,000 lux for 16 hr per day. host range of D. citri and the HLB Vector. About three hundred pathogen, under a cooperative adults of D. citri were collected from project between the Japan Interna- Murraya paniculata trees planted in tional Research Center for Agricul- the yard of Department of Agricul- 272 Thirteenth IOCV Conference, 1996-Procaryotes and Blight ture, Bangkok and reared for propa- symptoms and was severely stunted. gation on one-year-old seedlings of The rootstock developed suckers M. paniculata in the incubator. The that showed leaf mottling and yel- psylla laid eggs and increased abun- lowing, and partial die-back; and the dantly. The seedlings were replaced budunion was swollen. Midveins of every 2 to 3 mo with vigorous ones. the young leaves of the L. acidissima Young psylla adults were trans- that showed HLB-like symptoms ferred to the HLB-source plants and were collected in March 1995. They maintained for at least 1 wk in a were fixed in glutaraldehyde, post- plastic cage to acquire the HLB fixed with osmium and then embed- organism. The minimum acquisition ded into epoxy resin. Ultrathin sec- period of psylla being 1to 2 days (9). tions were made with a Sorvall MT2- Seedlings used for the experi- B ultramicrotome using a diamond ment. Seeds of the rutaceous plants knife and observed with an Hitachi were obtained from USDA-National H-300 electron microscope after dou- Clonal Germplasm Repository, Riv- ble staining with uranyl acetate and erside, California in December lead nitrate. 1992, except for Poncirus trifoliata, M. paniculata and L. acidissima. RESULTS Trifoliate orange was from Japan and the others were from Thailand. Population trends of D. citri Of the 20 species of the Rutaceae on Rutaceae plants. The popula- acquired, seeds of 12 species germi- tion trends of psylla on Clausena nated. Each plant was individually lansium, M. paniculata, local man- potted and grown in an insect-proof darin Som-keo-wan and L. acidis- screenhouse. Insecticide application sima varied markedly in the three was stopped 1 mo before vector repetitions. Some instances of transmission. decrease were caused by fungal Vector transmission and pop- infection because of high humidity. ulation trends of psylla. Fifteen Large numbers of psylla were alive psylla were transferred from the for the first week after transfer. donors to each receptor plant which Marked increase was noted 4 wk was covered with a net cage in an after transfer on the following incubator. The number of adults on plants: M. paniculata, Atalantia sp., the plants were counted at weekly and L. acidissima (Table 1). On intervals for 5 to 7 wk. After killing those plants, the psylla laid eggs the psylla by insecticide spray, the which gave rise to abundant devel- plants were transferred to a screen- opment of nymphs and adults. house for further observation of Small increases in the number of symptoms. The experiments were adults were observed on Murraya done from August to October 1993 koenigii and Som-keo-wan. No with three repetitions. As a control, increase, but a long term survival of two healthy seedlings were used. more than 7 wk, was noted on C. The plants were cut back in Decem- lansium (one out of three), Severinia ber and subsequent growth and buxifolia, Poncirus trifoliata, Bal- symptom development were samocitrus dawei and Som-pan observed until March 1994. mandarin. The survival of more than Electron microscopy. Electron 5 wk was noted on Triphasia trifolia, microscopy was conducted on L. Microcitrus australasica, Aeglopsis acidissima used as a rootstock in the chevalieri and Ladu mandarin. In field of Pichit Horticultural Research contrast, a rapid death of psylla was Center. Approximately 5-yr-old observed on C. lansium (two out of sweet orange, Som-tra, on the L. three) and Eremocitrus glauca acidissima showed typical HLB (Table 1). m TABLE 1 SURVIVAL AND MULTIPLICATION OF DIAPHORINA CITRI ON RUTACEAE PLANTS MAINTAINED IN A CAGE AND DEVELOPMENT OF DISEASE $. SYMPTOMS 8 No. adult psylla 2 s Exp. 1(wk) Exp. 2 (wk) Exp. 3 (wk) Symptoms B 2 Plant used for rearing psylla 1345 134567 134567 Leaf-mottle Stunting 83 k Clausena Iansium 8100 1100000 11118853 Questionable CD V, Murraya paniculata 12 9 48~51 14 9 lly 10 9 @ None o, I M. koenigii 14 12 12 147 14 14 13 13 10 3 9 9 8 10y 5 3. None 'u Triphasia trifolia 10522 1441100 None ++ 8 Severinina buxifolia 12 12 7 6 12 9 9 6 1 0 11 13 13 13 12 8 None + Atalantia sp. 12 13 48~52 37 37 None 2W Eremocitrus glauca 8100 Death Microcitrus australasica 12988 None + R Poncirus trifoliata 15 12 12 12 14 13 13 8 6 5 15 15 15 14 14 10 Questionable R Citrus reticulata !3 "Ladu" 148 54 1322100 1574400 Positive $ "Som-pan" 1476531 14133110 Death "Som-keo-wan" 14 6 5 18~ 13 14 5 4 3 3' 13 13 15~6 3 3 Questionable Balsamocitrus dawei 14 12 9 3 14 14 13 10 10 10 10 10 7 3 2 0 Questionable Aeglopsis chevalieri 14 10 10 9 _W Limonia acidisimma (= Ferronia limonia) 12 8 4 77 14 12 39y 42 53 46 15 16 30~39 43 41 Positive Fifteen adult psyllids were transferred to each plant. The cage was kept at 28126°C (daylnight) with 16 hr illumination in an incubator. .Killed by fungal infection increase in psylla .Yellowing of leaf followed by defoliation -No data because of no leaves '++ = severe stunting; + = moderate stunting; - = no stunting. 274 Thirteenth ZOCV Conference, 1996-Procaryotes and Blight Symptom development after paniculata, L. acidissima and Atal- vector transmission. Typical antia sp. In terms of feeding, egg lay- symptoms such as leaf mottle were ing, and nymph and adult observed in seedlings of Ladu man- development, there was no differ- darin and L. acidissima (Table 1).C. ence between L. acidissima and M. lansium, P. trifoliata, Som-keo-wan paniculata. Because L. acidissima and B. dawei showed mild mottling is a popular local fruit in Thailand, but still questionable as HLB symp- further observation in the fields is toms. Trifoliate orange and Som-pan needed to assess its role in HLB epi- mandarin died, but it was not deter- demiology. mined whether this was caused by Some population increase of D. HLB. Stunting was noted on T trifo- citri was observed on M. koenigii. In lia, S. buxifolia and M. australasica. West Malaysia, this was found to be This data were obtained in mid a common alternate host of D. citri March, after which high tempera- (6). We did not observe psylla multi- tures caused plant injury.