Michael N. Pearson

Mycoviruses of filamentous fungi and their relevance to plant pathology

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal-virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.

Morphological and molecular analysis of Colletotrichum acutatum sensu lato

The genetic relationships between several morphological groups recognized within Colletotrichum acutatum sensu lato were investigated using RAPD analysis and vegetative compatibility analysis. Isolates were examined from fruit rots originating in New Zealand and Australia, from Lupinus spp. in New Zealand, Canada, France and the U.K., and from Pinus radiata in New Zealand and Australia. The genetic distinctness of the groups recognized morphologically is confirmed. Two genetically distinct groups of C. acutatum-like pathogens are recognized from lupin, one comprising isolates from New Zealand and the U.K., the other isolates from Canada and France. C. acutatum f. sp. pineum isolates from New Zealand and Australia form two genetically distinct groups.

Identification and Validation of Reference Genes for Normalization of Transcripts from Virus-Infected Arabidopsis thaliana

Real-time quantitative polymerase chain reaction (qPCR) of complementary DNA is now a standard method for studies of gene expression. However, qPCR can identify genuine variation only when transcript quantities are accurately normalized to an appropriate reference. To identify the most reliable reference genes for transcript quantification by qPCR, we describe a systematic evaluation of candidate reference genes of Arabidopsis thaliana ecotype Columbia-0 (Col-0). Twelve genes were selected for transcript stability studies by qPCR of complementary DNA prepared from Arabidopsis leaf tissue infected with one of five plant viruses (Cauliflower mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, Turnip mosaic virus, and Turnip yellow mosaic virus). The F-box family protein, elongation factor 1-α, sand family protein, and protodermal factor 2 gene transcripts showed the most stable accumulation, whereas a traditionally used reference gene, Actin8, showed the least stable accumulation as measured by the geNorm algorithm. The data furnish plant virologists with reference genes for normalization of qPCR-derived gene expression in virus-infected Arabidopsis and will be beneficial to the selection and design of primers targeting orthologous genes in other plant species.

Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant ‘potex-like’ viruses

Summary.This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3′ terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant ‘potex-like’ viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant ‘potex-like’ coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant ‘potex-like’ virus group, distinct from BVF.

Presence of double-stranded RNA and virus-like particles in Botrytis cinerea

Double-stranded RNA (dsRNA) was demonstrated in mycelial extracts in 143 of 200 isolates of Botrytis cinerea from a range of hosts in New Zealand. The dsRNA profiles differed widely between isolates in the number, size and relative concentration of dsRNA species present. Virus-like particles (VLPs) were observed by electron microscopy in partly purified virus preparations in three of five dsRNA-containing isolates examined. Morphological types present included isometric particles of varying size classes (approx. 30, 35, 40 and 45 nm) and bacilliform particles (approx. 25 × 63 nm). Such particles were not observed in five isolates apparently lacking dsRNA but flexuous rods with a modal length of approx. 720 nm were present in one. Total RNA extraction of partly purified VLP preparations gave similar profiles to those obtained from mycelial dsRNA extractions of the same isolates, suggesting that the VLPs represent encapsidated dsRNA. Comparison of 12 dsRNA-containing and 12 dsRNA-free isolates indicated slight differences between the two groups in radial growth rate, virulence on bean leaves and sclerotial number but the differences were minor and the ranges overlapped.

Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain

The economically important rootstock species Poncirus trifoliata is resistant to most isolates of Citrus tristeza virus (CTV), but not to members of the CTV resistance-breaking (RB) strain presently found in New Zealand. In this study, five known and suspected RB isolates were separated from field mixtures, and their genomes were sequenced in full. It was found that the RB isolates are members of a single phylogenetically distinct clade with an average of 90.3% genomic nucleotide sequence identity to the closest extant isolate, T36. These isolates also show evidence of multiple recombination events throughout their evolutionary history, with T36, T30 and VT-like isolates, and with each other. Finally, the genomic sequences of these isolates show that several genes contain unique polymorphisms that may or may not be involved in overcoming resistance. These data will aid in the understanding of host–virus interactions, and the mechanism of resistance in P. trifoliata.

Plant pathogens transmitted by pollen

Pollen is a valuable source of germplasm for breeding and has few associated pests compared with other sources of genetic material. This review seeks to assist the development of appropriate phytosanitary measures by considering the pests that are transmitted by pollen. There are no invertebrates, bacteria, phytoplasmas or spiroplasmas that are pollen-transmitted. Only a limited number of fungal pathogens are associated with the pollen of a restricted number of hosts. In contrast, 39 viruses are pollen-transmitted and a further six are tentatively considered to be pollen-transmitted. The majority of these viruses belong to the Alphacryptovirus, Ilarvirus, Nepovirus or Potyvirus genera. Five viroids have also been identified as being pollen-transmitted.

A review of the plant virus, viroid and mollicute records for New Zealand

Considering New Zealand’s small size and remoteness, a remarkable number of plant pathogens have been recorded. A complete review of the records of plant viruses, viroids and mollicutes in New Zealand found good documentary evidence for 186 viral diseases, 5 viroids and 6 mollicutes, of which 59 viruses, 3 viroids, and 4 mollicutes have been recorded since publication of the previous complete review in 1989. Based on a lack of sufficient documented evidence, a further 27 viruses, 5 viroid and 3 mollicute records were considered unconfirmed. The confirmed pathogens not only infect agricultural and horticultural crops but some also affect the native flora.

Molecular characterization of three mitoviruses co-infecting a hypovirulent isolate of Sclerotinia sclerotiorum fungus.

Three double-stranded RNAs (dsRNAs) of 2438 nts (A), 2588 nts (B), and 2744 nts (C), from a single isolate of Sclerotinia sclerotiorum were sequenced. All three sequences showed similarity to known mitoviruses, consisting of a single open reading frame (ORF) with the characteristic conserved motifs of RNA-dependent RNA polymerase (RdRp). Mitochondrial malformations and reduced virulence and growth were associated with the presence of the dsRNAs. The terminal sequences of the (+) strand of the three dsRNAs could be folded into stem-loop structures and the inverted terminal complimentary sequences of dsRNA-A potentially form a panhandle structure. Sequence A showed 91.6% aa similarity to the previously described Sclerotinia sclerotiorum mitovirus 2 and was tentatively assigned the acronym SsMV2/NZ1. Sequences B and C showed only 16.4% similarity to each other and 15-48% aa similarity to the previously described mitoviruses and consequently appear to be new mitoviruses.

Nature, Incidence, and Symptomatology of Viruses Infecting Vanilla tahitensis in French Polynesia

A survey was carried out to identify the viruses infecting vanilla in French Polynesia and to assess their incidence. Virus identification was based on enzyme-linked immunosorbent assay (ELISA) and, for potyviruses, on the sequence of part of the coat protein and inoculation assays. Between 1998 and 1999, 3,610 vanilla plants from 49 plots in the Society Islands were indexed. Cymbidium mosaic virus (CymMV) was detected in 500 vines from 10 plots in the Leeward Islands. The data suggest that this virus has spread widely since its first detection in French Polynesia in 1986, most likely through the dissemination of symptomless infected cuttings. Viruses belonging to the Potyvirus genus were found in 674 plants from 27 plots in the four islands surveyed. Three distinct potyviruses have been identified: (i) Vanilla mosaic virus, (ii) Watermelon mosaic virus, and (iii) and a virus related to Bean common mosaic virus. The symptoms induced on Vanilla tahitensis by the three potyviruses can be differentiated from each other and from those due to CymMV. A significant proportion of the plants surveyed (97/476) were symptomatic but tested negative by ELISA for CymMV and the Potyvirus group. Odontoglossum ringspot virus was not detected in any sample tested.