BACKGROUND Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1-21. However, the pathogen elimination and colonization potential of B. subtilis L1-21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown. RESULTS Initially, we suggested that endophyte L1-21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium-Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1-21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1-21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1-21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively. CONCLUSIONS We propose that B. subtilis L1-21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP.

Huanglongbing (HLB) has turned into a devastating botanical pandemic of citrus crops, caused by Candidatus Liberibacter asiaticus (CLas). However, until now the disease has remained incurable with very limited control strategies available. Restoration of the affected microbiomes in the diseased host through the introduction of an indigenous endophyte Bacillus subtilis L1-21 isolated from healthy citrus may provide an innovative approach for disease management. A novel half-leaf method was developed in vitro to test the efficacy of the endophyte L1-21 against CLas. Application of B. subtilis L1-21 at 104 colony forming unit (cfu ml-1) resulted in a 1,000-fold reduction in the CLas copies per gram of leaf midrib (107 to 104) in 4 days. In HLB-affected citrus orchards over a period of 2 years, the CLas incidence was reduced to < 3%, and CLas copies declined from 109 to 104 g-1 of diseased leaf midribs in the endophyte L1-21 treated trees. Reduction in disease incidence may corroborate a direct or an indirect biocontrol effect of the endophytes as red fluorescent protein-labeled B. subtilis L1-21 colonized and shared niche (phloem) with CLas. This is the first large-scale study for establishing a sustainable HLB control strategy through citrus endophytic microbiome restructuring using an indigenous endophyte.

Huanglongbing (HLB), also known as citrus greening, is the most severe pandemics in citrus in more than 50 countries in Asia, Africa, and America, causing serious economic losses worldwide (Gottwald,2020 2020; Wang, 2019). The disease is associated with a phloem-limited and fastidious member of the α-proteobacteriacea. ‘Candidatus Liberibacter asiaticus’ (CLas) is the most prevalent strain. It was frequently detected in leaves, stems, and roots based on PCR detection, which recognized both living and dead cells. Yet only intact and viable CLas cells are potentially infectious and transmissible. Previous study showed 17 to 31% of CLas cells were considered viable in HLB symptomatic tissues (Trivedi et al., 2009). Whether viable CLas is present in floral organs remains unclear and the possibility that the embryo could be infected via pollen has not yet been addressed. In this study, we aimed to identify viable CLas in citrus floral parts and the possibility of the dissemination through pollination process with anti-OmpA and anti-SDE1, two highly specific antibodies against CLas (Ding et al., 2020; Tran et al., 2020). These results will deepen our knowledge of the distribution in planta and the new dissemination pathway of CLas, which is important for the monitoring of HLB. The study was carried out with 1113 samples of 5 citrus varieties collected in China. CLas DNA was detected in anther filament, pollen grains, stigma, ovary, and receptacle (Figure 1A). Significant differences were observed among the detection ratio in different floral organs (P < 0.05). Direct tissue blot immuno assay (DTBIA) revealed purple colour in the tissues from CLas-infected stigmas, ovary, locules, and receptacle (Figure 1B). And in developing anthers, pollen grains, and the germinated pollen tubes (Figure 1C). We expected that the invasion of viable CLas into floral parts would follow the same as the invasion of branch buds. Unpollinated lemon flower buds were tested. CLas was located in the phloem of both the branch and flower bud, especially in the joint connection between the receptacle and flower branch (Figure 1D). Therefore, the early infection of flower buds was originated from the mother plant through phloem connections. To find out the possibility of CLas dissemination from stigma to ovary, artificial cross-pollination was performed (Figure 1F). In paternal pollens, signals were observed inside the pollen grains in the locules as well as in the vascular bundles. After cross-pollination, CLas were firstly detected on the stigma in 4 hpp, and then moved from stigma to stylet via pollen tubes. In 3 dpp, CLas was found in the ovary and mainly localized in the ovary wall, as well as in locules. These results coincided with PCR (Figure 1E) and were consistent with the germination rate of CLas-affected pollen tubes observed by fluorescence microscopy (Figure 1G). Our results showed the dissemination of CLas from stigma to ovary through pollination process. In this study, the presence of viable CLas in the pollen itself, especially in the pollen tube, opens the possibility that infection of the embryo could occur from the pollen and thus avoid the chalazal barrier. Based on our data, the distribution of CLas within an infected flower and a pathway for the potential infection of the ovary of a healthy flower can be summarized (Figure 1H). It is worth noting that seedlings grew out from CLas-affected fertile seeds indicated the existence of the bacteria in extreme low titre and became undetectable in the late stage (data not shown). We suggest that the aborted seed may be the result of infection of the embryo via pollination, whereas the viable seed with contaminated seed coats or rarely embryo are largely due to the infection from the maternal plant. The low title of CLas infection failed to sustain itself as the seedlings grew, suggesting that the CLas populations being transmitted to the seedling either not existed in a viable form or missed some of the populations necessary for multiplication and virulence. In conclusion, viable CLas was firstly identified in citrus floral organs, especially in pollen and pollen tubes. The distribution of CLas in pollen grains and pollen tubes opened a new possible dissemination pathway through pollination which should be also taken into account for the integrated control of HLB. This study was supported by the Science and Technology Major Project of Guangxi (Gui Ke AA18118046), the National Natural Science Foundation of China (31872077), the National Key Research & Development Program of China (2018YFD0201500), and the Fundamental Research Funds for the Central Universities (2662016PY099). The authors declare no competing interests. Q.L. Wang, Y.L. Xu, and X.F. Yang initiated the study, and contributed equally; J. Jia, J.L. Zhou, J.W. Zeng, X. Yan, J.X. Li, J.Q Yue, J. Guo, Y. Yang, C.X. Xia contributed samples and data analysis. N. Hong, G.P. Wang, S.A. Peng, Y.P. Duan, J.S. Hartung, and F. Ding contributed to critically revising of the manuscript.

BACKGROUND:In insects, little is known about the co-evolution between their primary endosymbionts and hosts at the intraspecific level. This study examined co-diversification between the notorious agricultural pest Diaphorina citri and its primary endosymbionts (P-endosymbiont), 'Candidatus Carsonella ruddii' at the population level.RESULTS:Maximum likelihood, haplotype network, principal components and Bayesian clustering identified three lineages for D. citri and its P-endosymbiont: a Western clade containing individuals from Pakistan, Bhutan (Phuentsholing), Vietnam (Son La), USA, Myanmar and China (Ruili, Yunnan); a Central clade, with accessions originating from Southwest China, Bhutan (Tsirang) and Bangladesh; and an Eastern clade containing individuals from Southeast Asia, and East and South China. A more diverse genetic structure was apparent in the host mitochondrial DNA than their P-endosymbionts; however, the two sets of data were strongly congruent.CONCLUSION:This study provides evidence for the co-diversification of D. citri and its P-endosymbiont during the migration from South Asia to East and Southeast Asia. We also suggest that the P-endosymbiont may facilitate investigations into the genealogy and migration history of the host. The biogeography of D. citri and its P-endosymbiont indicated that D. citri colonized and underwent a secondary dispersal from South Asia to East and Southeast Asia. © 2018 Society of Chemical Industry.

[Objective]The purpose of the study was to find out the arthropod species in lemon orchard of Dehong Prefecture and to provide scientific references for cumulative control on limiting lemon pests.[Method]From June 2006 to July 2009,arthropod species in 19 lemon orchards from Dehong-lemon-production area were investigated in every season for a year using visual observation method and net capturing method.[Result]The results showed that the arthropods have 2 classes,11 orders,50 families,and 92 species,among which detrimental insects have 2 classes,8 orders,30 families,and 57 species;beneficial insects have 2 classes,6 orders,6 families,and 29 species;lastly,neutral insects have 2 classes,2 orders,6 families,and 6 species.The number of families and species of homoptera occupied the largest proportion of the total number of families and species of injurious insects,which were 30.00% and 35.09%,respectively.The number of families and species for hymenoptera occupied the largest proportion in the total number of families and species of beneficial insects,which were 43.75% and 34.48%,respectively.Diptera and blattaria were the dominant species of neutral insects,and the number of families and species of diptera held the maximum ratio,in which both categories were as high as 83.33%.[Conclusion]Among the many kinds of arthropod in Dehong lemon orchards,detrimental insects were the dominant type of pests in terms of population number.Beneficial insects also had many species,but their quantity was too low to effectively control pests,so chemical control should be applied in lemon orchard.

Nymphs of the pomelo psyllid, Cacopsylla (Psylla) citrisuga Yang & Li, were collected from huanglongbing (HLB) symptomatic lemon trees, Citrus limon (L.) in Yunnan Province, China. DNA samples extracted from groups of 2-10 pomelo psyllid nymphs of all stages and from leaves of lemon plants were analyzed with nested-PCR in order to detect 'Candidatus Liberibacter asiaticus' (Las). The results showed that 24 out of the 49 lemon trees were Las-positive. Also psyllid nymphs collected from 10 out of the 24 Las-positive plants were Las-positive. DNA extracted from individual late stage nymphs (3rd to 5th instars) collected from Las-infected trees were then subjected to nested-PCR trials. Twelve out of the 29 nymphs proved to be Las positive. All nymphs collected from Las-negative lemon trees were also Las-negative. Most psyllid samples which showed positive in nested-PCR were also positive in conventional-PCR detection. The amplified fragment of the 16S rRNA gene of 'Ca. Liberibacter spp.' from positive psyllid samples was 99% similar to those of Las strain psy62 in GenBank. These results demonstrate that Cacopsylla (Psylla) citrisuga is another Las carrier insect. Transmission studies are underway to determine whether Las-positive psyllids can transmit Las to healthy citrus.