Biochemical and molecular tools reveal two diverse Xanthomonas groups in bananas.

Xanthomonas campestris pv. musacearum (Xcm) causing the banana Xanthomonas wilt (BXW) disease has been the main xanthomonad associated with bananas in East and Central Africa based on phenotypic and biochemical characteristics. However, biochemical methods cannot effectively distinguish between pathogenic and non-pathogenic xanthomonads. In this study, gram-negative and yellow-pigmented mucoid bacteria were isolated from BXW symptomatic and symptomless bananas collected from different parts of Uganda. Biolog, Xcm-specific (GspDm), Xanthomonas vasicola species-specific (NZ085) and Xanthomonas genus-specific (X1623) primers in PCR, and sequencing of ITS region were used to identify and characterize the isolates. Biolog tests revealed several isolates as xanthomonads. The GspDm and NZ085 primers accurately identified three isolates from diseased bananas as Xcm and these were pathogenic when re-inoculated into bananas. DNA from more isolates than those amplified by GspDm and NZ085 primers were amplified by the X1623 primers implying they are xanthomonads, these were however non-pathogenic on bananas. In the 16-23 ITS sequence based phylogeny, the pathogenic bacteria clustered together with the Xcm reference strain, while the non-pathogenic xanthomonads isolated from both BXW symptomatic and symptomless bananas clustered with group I xanthomonads. The findings reveal dynamic Xanthomonas populations in bananas, which can easily be misrepresented by only using phenotyping and biochemical tests. A combination of tools provides the most accurate identity and characterization of these plant associated bacteria. The interactions between the pathogenic and non-pathogenic xanthomonads in bananas may pave way to understanding effect of microbial interactions on BXW disease development and offer clues to biocontrol of Xcm.

Characterization and Distribution of a Potyvirus Associated with Passion Fruit Woodiness Disease in Uganda.

This article describes the incidence and etiology of a viral disease of passion fruit in Uganda. Symptoms, including those characteristic of passion fruit woodiness disease (PWD), were observed on 32% of plants in producing areas. Electron microscopic observations of infected tissues revealed flexuous filaments of ca. 780 nm. Enzymelinked immunosorbent assays indicated a serological relationship with Cowpea aphid-borne mosaic virus (CABMV) and Passion fruit ringspot virus (PFRSV). In host range studies, only species in the families Solanaceae and Chenopodiaceae were susceptible, and neither Vigna unguiculata nor Phaseolus vulgaris became infected. Coat protein (CP) gene sequences of eight isolates exhibited features typical of potyviruses and were highly similar (88 to 100% identity). However, the sequences had limited sequence identity with CP genes of two of the three potyviruses reported to cause PWD: East Asian Passiflora virus and Passion fruit woodiness virus (PWV). Deduced amino acid sequences for the CP of isolates from Uganda had highest identity with Bean common mosaic necrosis virus (BCMNV) (72 to 79%, with evolutionary divergence values between 0.17 and 0.19) and CABMV (73 to 76%, with divergence values between 0.21 and 0.25). Based on these results and in accordance with International Committee for Taxonomy of Viruses criteria for species demarcation in the family Potyviridae, we conclude that a previously unreported virus causes viral diseases on passion fruit in Uganda. The name "Ugandan Passiflora virus" is proposed for this virus.

Analysis of expressed sequence tags from Maize mosaic rhabdovirus-infected gut tissues of Peregrinus maidis reveals the presence of key components of insect innate immunity.

The corn planthopper, Peregrinus maidis, causes direct feeding damage to plants and transmits Maize mosaic rhabdovirus (MMV) in a persistent-propagative manner. MMV must cross several insect tissue layers for successful transmission to occur, and the gut serves as an important barrier for rhabdovirus transmission. In order to facilitate the identification of proteins that may interact with MMV either by facilitating acquisition or responding to virus infection, we generated and analysed the gut transcriptome of P. maidis. From two normalized cDNA libraries, we generated a P. maidis gut transcriptome composed of 20,771 expressed sequence tags (ESTs). Assembly of the sequences yielded 1860 contigs and 14,032 singletons, and biological roles were assigned to 5793 (36%). Comparison of P. maidis ESTs with other insect amino acid sequences revealed that P. maidis shares greatest sequence similarity with another hemipteran, the brown planthopper Nilaparvata lugens. We identified 202 P. maidis transcripts with putative homology to proteins associated with insect innate immunity, including those implicated in the Toll, Imd, JAK/STAT, Jnk and the small-interfering RNA-mediated pathways. Sequence comparisons between our P. maidis gut EST collection and the currently available National Center for Biotechnology Information EST database collection for Ni. lugens revealed that a pathogen recognition receptor in the Imd pathway, peptidoglycan recognition protein-long class (PGRP-LC), is present in these two members of the family Delphacidae; however, these recognition receptors are lacking in the model hemipteran Acyrthosiphon pisum. In addition, we identified sequences in the P. maidis gut transcriptome that share significant amino acid sequence similarities with the rhabdovirus receptor molecule, acetylcholine receptor (AChR), found in other hosts. This EST analysis sheds new light on immune response pathways in hemipteran guts that will be useful for further dissecting innate defence response pathways to rhabdovirus infection.

Host Range, Purification, and Genetic Variability in Sweet potato chlorotic fleck virus.

Sweet potato chlorotic fleck virus (SPCFV) has recently been classified as a putative new member of the genus Carlavirus (family Flexiviridae) on the basis of its molecular properties. In this study, SPCFV was characterized in terms of host range, physical and biological characteristics, and genetic variability. In addition to sweet potato, SPCFV infected some plant species in the families Convolvulaceae, Chenopodiaceae, and Solanaceae. Limited numbers of virus particles were observed in the assimilation parenchyma cells of infected plant tissues; some cells had a distorted and enlarged endoplasmic reticulum though without any cytoplasmic and amorphous inclusions. The normal length of SPCFV particles was determined to be approximately 800 nm. In enzyme-linked immunosorbent assays, polyclonal antibodies raised against purified SPCFV virions were able to detect the virus in infected sweet potato and indicator plant tissues. In immunoelectron microscopy, SPCFV particles were all strongly decorated when reacted with homologous antiserum. Comparison of the 3' terminal part of the genome of a range of geographically diverse isolates revealed a high level of genetic diversity. The amino acid sequence identity in the coat protein and the nucleic acid binding protein ranged from 89 to 99.7% and from 75.9 to 99.2%, respectively. Phylogenetic analysis of both proteins showed a geographically associated clustering into two genogroups.

Sequence analysis of the entire RNA genome of a sweet potato chlorotic fleck virus isolate reveals that it belongs to a distinct carlavirus species.

Since the paucity of information on sweet potato chlorotic fleck virus (SPCFV) had precluded its classification, we have determined the complete nucleotide sequence of the single-stranded RNA genome of a Ugandan isolate of SPCFV. The genome is 9104 nucleotides long (excluding the poly(A) tail) and potentially includes six open reading frames (ORFs). Based on genomic organisation and sequence similarity, SPCFV appears to be a member of the genus Carlavirus (family Flexiviridae). However, SPCFV is distantly related to typical carlaviruses, as most of its putative gene products share amino acid sequence identities of <40% with those of typical carlaviruses. Its closest relative is melon yellowing-associated virus, a proposed carlavirus from Brazil, with which it shares ORF5 and ORF6 amino acid sequence identities of 61 and 46%, respectively.