The roles of psyllids, host plants and environment in the aetiology of huanglongbing in Bhutan

Abstract

'Candidatus Liberibacter asiaticus' ('CLas') (a-Proteobacteria) is the pathogen associated with the Asiatic form of huanglongbing (HLB), the most serious disease of citrus in Asia and the Americas, in Bhutan. The principal vector of the disease, Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), is also present in Bhutan. Recent detection of 'CLas' in the black psyllid, Diaphorina communis Mathur (Hemiptera: Liviidae), prompted my PhD study. I examined the molecular characteristics of Diaphorina communis and Diaphorina citri and found that the mitochondrial COI and 16S regions can distinguish the two species, and is useful for the identification of nymphs in the absence of adults and fifth instar nymphs. Investigations into whether Diaphorina communis can acquire 'CLas' and transmit it from 'CLas'-infected mandarin (Citrus reticulata Blanco) to healthy seedlings of mandarin and curry leaf (Bergera koenigii L.) and vice versa were conducted under field conditions at Baychhu (27.2975ºN, 89.9669ºE, 784 m ASL), Wangdue Phodrang Dzongkhag. Several experiments were conducted starting with preliminary experiments in 2013, and transmission experiments in 2014 and 2015. Results showed that Diaphorina communis did not reproduce on mandarin and preferred curry leaf to mandarin given the choice. Further, a low (~6%) proportion of Diaphorina communis acquired 'CLas' after 16-47 d and 39 d of acquisition and transmission intervals in 2014 and 2015, respectively. No transmission of 'CLas' from 'CLas'-infected mandarin to healthy mandarin and curry leaf by Diaphorina communis could be established based on periodic testing of mandarin and curry leaf seedlings. No transmission of 'CLas' from curry leaf to mandarin or curry leaf could be achieved. Phylogenetic analyses based on ITS 1 and 2, and the COI region as well as the morphological descriptions revealed that the individuals of the parasitoid reared from parasitised nymphs of Diaphorina communis belonged to a new species of Tamarixia. The name of the new species has been proposed as Tamarixia drukyulensis sp. n. (Hymenoptera: Eulophidae). Assessment of the effect of altitude, ambient temperature, relative humidity and rainfall on leaf temperatures of mandarin plants, HLB, and distribution of psyllids, was made using eight sites ranging from 800 m to 1500 m ASL along Droopchhugang-Phuensoomgang in Tsirang. My investigations revealed that, in contrast to my hypotheses, leaf temperatures were lower than ambient temperature at all elevations and did not influence the low incidences of 'CLas' and Diaphorina citri at higher elevations; mandarin seedlings grew better at 800-1000 m ASL than seedlings planted at 1100 and 1500 m ASL in spite of higher incidences of 'CLas' and Diaphorina citri at 800-1100 m ASL; ambient temperatures and relative humidity at all elevations were within the range of conditions favourable for development and survival of Diaphorina citri. UV radiation could have a role in the distribution of psyllids in addition to the interaction of rainfall and ambient temperatures at elevations = 1200 m ASL; Diaphorina communis were observed on Bergera koenigii at 800, 900 and 1000 m but not on mandarin seedlings in the experimental plots. Cacopsylla heterogena Li (Hemiptera: Sternorrhyncha: Psyllidae) was found on the mandarin seedlings from 1300 m to 1500 m ASL in the experimental plots in Tsirang. It was also recorded on citrus above and below this range in other locations. The molecular phylogenetic analyses using COI, 16S and ITS regions revealed that green psyllids on citrus belonged to one species, Cacopsylla heterogena. Two species of psyllids occurring on Zanthoxylum sp. were identified as Cornopsylla rotundiconis Lou, Li, Li & Cai and an unidentified Cacopsylla sp. (Hemiptera: Sternorrhyncha: Psyllidae). However, the molecular phylogenetic analyses could not resolve the separation of Cacopsylla heterogena from Cacopsylla citrisuga (Yang & Li), a species occurring in China. Real-time PCR analyses did not detect 'CLas' in Cornopsylla rotundiconis and the unidentified Cacopsylla sp. Only a small portion (~2%) of adults of individuals of Cacopsylla heterogena were 'CLas' positive despite most citrus plants from the same the locations where the insect was collected being positive for 'CLas'; no nymphs were 'CLas' positive. The insects on the Zanthoxylum sp. were regarded as not harbouring 'CLas'. The Murraya plants occurring in Baychhu, Wangdue Phodrang and in Reldri along Rinchending-Pasakha at Phuentsholing (26.8400ºN, 89.4045ºE, 400 m ASL) were identified as Murraya elongata A. DC. ex Hook. f. based on molecular analyses using five cpDNA (matK-5'trnK spacer, psbM-trnDGUC spacer; rpS4-trnT spacer, trnCGCA-ycf6 region trnL-trnL-trnF region) and the ITS region. Analyses of morphological features of leaves also separated it from Murraya paniculata (L.), and Murraya heptaphylla Spanoghe but not from Murraya sumatrana Roxburgh. Morphological differentiation between Murraya elongata and Murraya sumatrana was, therefore, based on the shapes of fruit and seeds. Adults of both Diaphorina communis and Diaphorina citri were observed on Murraya elongata in Baychhu but the eggs and nymphs occurring on these plants were confirmed as those of Diaphorina citri. No confirmation of the presence of 'CLas' in Murraya elongata could be established. The identity of a wild citrus species observed at near Wengkhar in Mongar and at Basochhu in Wangdue Phodrang could not be resolved based on four cpDNA and ITS, and morphological descriptions. Although, leaf damage and nymphs of Cacopsylla heterogena were observed on these wild citrus, leaf samples tested for 'CLas' did not indicate any presence of the pathogen. The study provides information on the role of other citrus-feeding psyllids and host plants that could be involved in the transmission of HLB in Asia. It has implications for management of HLB and citrus production in Bhutan and other countries with similar situations. Based on my results, it should be feasible to grow citrus above 1200 m ASL if suitable varieties can be found and if powdery mildew is controlled. Citrus production below 1200 m should also be feasible if all citrus below 1200 m in Bhutan is progressively removed and replanted at high densities with certified pathogen-free trees under strict quarantine.

Date of Award	2017
Original language	English