Hong-Ji Su

Reassortment and Concerted Evolution in Banana Bunchy Top Virus Genomes

ABSTRACT The nanovirus Banana bunchy top virus (BBTV) has six standard components in its genome and occasionally contains components encoding additional Rep (replication initiation protein) genes. Phylogenetic network analysis of coding sequences of DNA 1 and 3 confirmed the two major groups of BBTV, a Pacific and an Asian group, but show evidence of web-like phylogenies for some genes. Phylogenetic analysis of 102 major common regions (CR-Ms) from all six components showed a possible concerted evolution within the Pacific group, which is likely due to recombination in this region. The CR-M of additional Rep genes is close to that of DNA 1 and 2. Comparison of tree topologies constructed with DNA 1 and DNA 3 coding sequences of 14 BBTV isolates showed distinct phylogenetic histories based on Kishino-Hasegawa and Shimodaira-Hasegawa tests. The results of principal component analysis of amino acid and codon usages indicate that DNA 1 and 3 have a codon bias different from that of all other genes of nanoviruses, including all currently known additional Rep genes of BBTV, which suggests a possible ancient genome reassortment event between distinctive nanoviruses.

Unusual Events Involved in Banana bunchy top virus Strain Evolution

Banana bunchy top virus (BBTV) can be transmitted by aphids and consists of at least six integral components (DNA-R, -U3, -S, -M, -C, and -N). Several additional replication-competent components (additional Reps) are associated with some BBTV isolates. A collected BBTV strain (TW3) that causes mild symptoms was selected to study the processes in BBTV evolution. Southern blot hybridization, polymerase chain reaction (PCR), and real-time PCR did not detect DNA-N in TW3. Real-time PCR quantification of BBTV components revealed that, except for the copy number of TW3 DNA-U3, each detected integral component of BBTV TW3 was at least two orders lower than that of the severe strains. No infection was observed in plants inoculated with aphids, which were first given acquisition access to the TW3-infected banana leaves. Recombination analysis revealed recombination between the integral component TW3 DNA-U3 and the additional Rep DNA-Y. All BBTV integral components contain a replication initiation region (stem-loop common region) that share high sequence identity. Sequence alignment revealed that TW3 DNA-R, -S, -M, and -C all have a stem-loop common region containing a characteristic 9-nucleotide deletion found only in all reported DNA-N. Our data suggest that the additional Rep DNAs can serve as sources of additional genetic diversity for integral BBTV components.

Identification of the Chinese Box Orange (Severinia Buxifolia) as an Alternative Host of the Bacterium Causing Citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2–3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3–4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBB-free psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.

Interaction between Likubin Bacterium and Phytophthora parasitica in Citrus Hosts

Infection with Likubin bacterium (LB) followed by Phytophthora parasitica increased the mortality of sour orange and pummelo seedlings, and enhanced the P. parasitica-induced root rot in all the four types of citrus tested. The LB-induced enhancement of root infection by P. parasitica was apparent within 1 h of exposure to zoospore suspension. The enhancement of P. parasitica-induced root rot was affected by the infection sequence. Inoculation of sour orange seedlings with LB before P. parasitica was more effective in increasing P. parasitica-induced root rot than LB and P. parasitica concomitantly or LB after P. parasitica. Grafting P. parasitica susceptible scions of ponkan (Citrus reticulata) onto P. parasitica-tolerant rootstocks of sour orange greatly increased the susceptibility of rootstocks to P. parasitica. Results also demonstrate the enhancement of LB-induced symptoms by P. parasitica in citrus plants.

Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant.

Abstract Little is known about how plant viruses of a single species exhibit different movement behavior in different host species. Two Cymbidium mosaic potexvirus (CymMV) isolates, M1 and M2, were studied. Both can infect Phalaenopsis orchids, but only M1 can systemically infect Nicotiana benthamiana plants. Protoplast inoculation and whole-mount in situ hybridization revealed that both isolates can replicate in N. benthamiana; however, M2 was restricted to the initially infected cells. Genome shuffling between M1 and M2 revealed that two control modes are involved in CymMV host dependent movement. The M1 coat protein (CP) plays a dominant role in controlling CymMV movement between cells, because all chimeric CymMV viruses containing the M1 CP systemically infected N. benthamiana plants. Without the M1 CP, one chimeric virus containing the combination of the M1 triple gene block proteins (TGBps), the M2 5′ RNA (1–4333), and the M2 CP effectively moved in N. benthamiana plants. Further complementation analysis revealed that M1 TGBp1 and TGBp3 are co-required to complement the movement of the chimeric viruses in N. benthamiana. The amino acids within the CP, TGBp1 and TGBp3 which are required or important for CymMV M2 movement in N. benthamiana plants were mapped. The required amino acids within the CP map to the predicted RNA binding domain. RNA–protein binding assays revealed that M1 CP has higher RNA binding affinity than does M2 CP. Yeast two-hybrid assays to detect all possible interactions of M1 TGBps and CP, and only TGBp1 and CP self-interactions were observed.

Cochin China atalantia (Atalantia citroides) as a new alternative host of the bacterium causing citrus Huanglongbing

Through graft inoculation and psyllid transmission experiments, the Cochin China atalantia (Atalantia citroides) was found to be a new alternative host of the fastidious bacterium (Candidatus Liberibacter asiaticus = Las) causing citrus Huanglongbing (HLB). The Las pathogen is commonly transmitted by Asian citrus psyllid (Diaphorina citri) in persistent manner. The Cochin China atalantia plants locally called wild lime (WL) including elliptic leaf (WL-1) and elongated leaf (WL-2) types are growing wildly in Cambodia and Cochin China of Southern Vietnam. The Las pathogen infected Cochin China atalantia plants by graft inoculation and psyllid transmission, and survived persistently. The Las was transmitted from Las-infected Cochin China atalantia plants of two types to healthy citrus plants (Ponkan mandarin) and atalantia plants through psyllid transmission. Therefore, the Cochin China atalantia plants of two types may serve as a new alternative host of Las. Higher titer of Las was detected in the Las-infected WL-1 atalantia plant which developed typical yellow mottling symptom, while lower titer of Las was detected in WL-2 atalantia plant developing mild chlorosis symptom.

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts, rootstocks and the Asian citrus psyllid

Using proper managements for Citrus Huanglongbing (HLB), which caused by Candidatus Liberibacter asiaticus (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. The HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP population. We studied dynamic populations of Las in different citrus hosts and each instar of ACP and also examined the seasonal growth and distribution of Las in different tissues using conventional and TaqMan® real-time PCR. The results showed obvious symptoms and poor growth on hosts or rootstocks, indicating different levels of susceptibility/tolerance to HLB. Troyer citrange, Swingle citrumelo and woody apple are highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis, mature and old leaves contained high concentrations of Las at cool temperatures in autumn and spring. For periodic detection, Las were detected earlier through psyllid-transmission than through graft-inoculation at 21 days post-infection, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (104-107 copy numbers/μL) and Las-carrying percentages (LCP) (40-53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP on psyllids were not affected by the increasing acquisition-access time. The present study has significant implications for disease ecology. The combination of early detection, using proper rootstocks and constraining psyllid populations could achieve better management of HLB disease. This article is protected by copyright. All rights reserved.

First Report of Banana Streak Virus Infecting Banana Cultivars (Musa spp.) in Taiwan

Banana (Musa sapientam L.) is an economically important crop for both export and local consumption in Taiwan. Recently, leaf symptoms characteristic of banana streak disease (1) were found on banana cv. Mysore (AAB group) introduced from Australia in the germ plasm collection of the Taiwan Banana Research Institute. The citrus mealybug (Planococus citri) has been shown to transmit banana streak virus (BSV) but not banana bunchy top virus or cucumber mosaic virus (CMV) (2). When mealybugs were fed on leaves of diseased Mysore banana and transferred to healthy banana cv. Cavendish seedlings in a growth chamber, the latter developed fine chlorotic streaks characteristic of symptoms caused by BSV within 1 to 3 months. Some chlorotic streaks became necrotic. BSV was detected in diseased but not healthy leaves of Mysore and Cavendish bananas by polymerase chain reaction (PCR) with primer pairs of BSV provided by J. E. Thomas of Queensland Department of Primary Industries. Subsequently, fine chlorotic streaks were observed in leaves of Cavendish banana in several fields in southern Taiwan. Some of these diseased plants developed severe leaf necrosis, causing heart rot of spindle leaves characteristic of symptoms caused by CMV. Presence of BSV in these plants was verified by PCR assay. However, CMV was also detected by double antibody sandwich-enzyme-linked immunosorbent assay with a monoclonal antibody to CMV, indicating that these plants were simultaneously infected by both viruses. This is the first report of BSV infecting Musa spp. in Taiwan. References: (1) B. E. L. Lockhart. Phytopathology 76:995, 1986. (2) B. E. L. Lockhart. 1995 Food & Fertilizer Technol. Center (ASPAC) Tech. Bull. 143. 11 pp.