The use of attenuated isolates of Pepino mosaic virus for cross-protection

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits

Evolutionary relationships between Rhynchosporium lolii sp. nov. and other Rhynchosporium species on grass.

Copyright: 2013 King et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThe fungal genus Rhynchosporium (causative agent of leaf blotch) contains several host-specialised species, including R. commune (colonising barley and brome-grass), R. agropyri (couch-grass), R. secalis (rye and triticale) and the more distantly related R. orthosporum (cocksfoot). This study used molecular fingerprinting, multilocus DNA sequence data, conidial morphology, host range tests and scanning electron microscopy to investigate the relationship between Rhynchosporium species on ryegrasses, both economically important forage grasses and common wild grasses in many cereal growing areas, and other plant species. Two different types of Rhynchosporium were found on ryegrasses in the UK. Firstly, there were isolates of R. commune that were pathogenic to both barley and Italian ryegrass. Secondly, there were isolates of a new species, here named R. lolii, that were pathogenic only to ryegrass species. R. lolii was most closely related to R. orthosporum, but exhibited clear molecular, morphological and host range differences. The species was estimated to have diverged from R. orthosporum ca. 5735 years before the present. The colonisation strategy of all of the different Rhynchosporium species involved extensive hyphal growth in the sub-cuticular regions of the leaves. Finally, new species-specific PCR diagnostic tests were developed that could distinguish between these five closely related Rhynchosporium species.Peer reviewedFinal Published versio

Characterisation and detection of dasheen mosaic potyvirus in Zantedeschia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Horticultural Science in Plant Health at Massey University, Palmerston North, New Zealand

Four potyvirus isolates believed to be dasheen mosaic potyvirus, the most frequently occurring virus to infect members of the Araceae, were obtained from Caladium, Colocasia, Xanthosoma and Zantedeschia in world-wide locations. Properties of these isolates such as particle length, serological relatedness, electrophoretic mobility of coat proteins and genomic characteristics were compared. Serologically distinct strains of dasheen mosaic potyvirus were apparent amongst the isolates. The difference in the serological relationship was coupled with a variation in symptom expression. An isolate from Colocasia esculenta (L.) Schott was not serologically related to the other isolates. Further isolates from C. esculenta also exhibited no relationship. The modal length was different as well as the ability of complementary deoxyribonucleic acid, produced to the viral ribonucleic acid, to bind with some of the primers used in the polymerase chain reaction. This evidence led to the proposal that the isolate from C. esculenta was not dasheen mosaic potyvirus; this virus is tentatively named taro feathery mottle potyvirus. Cytoplasmic inclusion protein aggregates of dasheen mosaic potyvirus were purified from infected leaf tissue. SDS-polyacrylamide gel electrophoretic analysis of samples revealed a major band with an estimated molecular weight of 68,000 daltons. Such a band was absent from healthy tissue samples. The ATPase activity in samples from each purification step was determined by measuring the amount of [32P] released from the [λ-32P]ATP during incubation with the cytoplasmic inclusion protein. The level of ATPase activity in each sample showed a strong correlation with the amount of protein that was present. In a limited survey of commercial plantings twenty nine tubers grown for cutflower or tuber export were obtained from seven properties at different locations in New Zealand and grown on in a greenhouse. Each plant was indexed for virus infection. Electron microscopy revealed that plants from three of the properties contained 720nm flexuous rods. Samples from all but two plants tested positive to a potyvirus group antiserum using the enzyme-linked immunosorbent assay. The remaining two plants tested positive in microprecipitin and rapid immune electron microscopy tests to an antiserum prepared to a member of the carlavirus group. Particles from these plants were mechanically transmitted to Nicotiana tabacum 'Havana'. Rod-shaped particles of 300nm were observed in plants from four properties and tested positive to tobacco mosaic tobamovirus antiserum using a microprecipitin test. While inoculations to herbaceous indicators resulted in no symptoms, 300nm particles were observed in samples from the indicator plants. Tomato spotted wilt tospovirus, potato X potexvirus and cucumber mosaic cucumovirus, reported to infect Zantedeschia spp, were not detected

Occurrence and diversity of Xanthomonas campestris pv. campestris in vegetable brassica fields in Nepal

Black rot caused by Xanthomonas campestris pv. campestris was found in 28 sampled cabbage fields in five major cabbage-growing districts in Nepal in 2001 and in four cauliflower fields in two districts and a leaf mustard seed bed in 2003. Pathogenic X. campestris pv. campestris strains were obtained from 39 cabbage plants, 4 cauliflower plants, and 1 leaf mustard plant with typical lesions. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) using repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus, and BOX primers was used to assess the genetic diversity. Strains were also race typed using a differential series of Brassica spp. Cabbage strains belonged to five races (races 1, 4, 5, 6, and 7), with races 4, 1, and 6 the most common. All cauliflower strains were race 4 and the leaf mustard strain was race 6. A dendrogram derived from the combined rep-PCR profiles showed that the Nepalese X. campestris pv. campestris strains clustered separately from other Xanthomonas spp. and pathovars. Race 1 strains clustered together and strains of races 4, 5, and 6 were each split into at least two clusters. The presence of different races and the genetic variability of the pathogen should be considered when resistant cultivars are bred and introduced into regions in Nepal to control black rot of brassicas

Fungal endophytes in species of paspalum and bromus, occurrence and in vitro antagonistic activity assessment

The aims of this work were to study the occurrence of fungiassociated to Paspalum dilatatum, P. notatum, Bromus biebersteiniiand B. catharticus and to evaluate their potential as antagonisticagents against fungal pathogens. The genera Acremonium, Alternaria,Aspergillus, Curvularia, Fusarium, Penicillium and Stemphylium werethe most representative isolated fungi. The isolates of the genusAcremonium showed antagonistic activity against different putativepathogenic species tested in dual cultures. The results suggest that theinteractions and mechanisms of antagonism of the same strain varyaccording to the putative pathogenic strains to which they are facedto. The results encourage further studies to elucidate if the strainsisolated in this study are good candidates for biological control of latentpathogens in pastures. To accomplish this, future studies focused onthe analysis of the secondary metabolites produced by the antagonistsin the culture medium, as well as assays in planta will be conducted.Fil: García Lemos, Adriana. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Gil Cardeza, Maria Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Gomez, Elena del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Felitti, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentin

The symptom and genetic diversity of cassava brown streak viruses infecting cassava in East Africa

The genetic and symptom diversity of six virus isolates causing cassava brown streak disease (CBSD) in the endemic (Kenya, Mozambique, and Tanzania) and the recently affected epidemic areas (Uganda) of eastern Africa was studied. Five cassava varieties; Albert, Colombian, Ebwanateraka, TMS60444 (all susceptible) and Kiroba (tolerant) were graft inoculated with each isolate. Based on a number of parameters including the severity of leaf and root symptoms, and the extent of virus transmission by grafting, the viruses were classified as either severe or relatively mild. These results were further confirmed by the mechanical inoculation of 13 herbaceous hosts in which the virulent isolates caused plant death in Nicotiana clevelandii and N. benthamiana whereas the milder isolates did not. Phylogenetic analysis of complete coat protein gene sequences of these isolates together with sequences obtained from 14 other field-collected samples from Kenya and Zanzibar, and reference sequences grouped them into two distinct clusters, representing the two species of cassava brown streak viruses. Put together, these results did not suggest the association of a hypervirulent form of the virus with the current CBSD epidemic in Uganda. Identification of the severe and milder isolates, however, has further implications for disease management and quarantine requirements

Kirramyces destructans in Australia: biosecurity threat or elusive native pathogen?

Kirramyces destructans was first described in 1996 from north Sumatra, Indonesia, where it caused severe leaf and shoot blight on Eucalyptus grandis in nurseries and young plantations. Since then it has been reported in nurseries and plantations in Vietnam, Thailand and China, with its host range extending to include E. camaldulensis and E. urophylla. K. destructans has also been reported from native E. urophylla in East Timor and was considered a significant biosecurity threat to Australia’s native eucalypt forests and plantations. A study on the population diversity of K. destructans isolates throughout south-east Asia in which 8 gene regions were sequenced (four nuclear genes, one mitochondrial gene and three microsatellite markers) detected very low nucleotide polymorphism. This genetic uniformity is indicative of an introduced population which has subsequently spread throughout Asia via human-mediated movement of germplasm. Surveys of sentinel plantings in northern Australia revealed a complex of Kirramyces spp. among which K. destructans was detected. The same gene regions and markers were sequenced as for the Asian study and diversity among the K. destructans isolates in Australia was found to be much greater than that in Asia. We believe that K. destructans is native to Australia where is resides symptomlessly within the native vegetation. The disease is only expressed when non-endemic eucalypts are planted. As such the pathogen is a major encumbrance to the establishment of commercial eucalypt plantations in Northern Australia. The disease has not been observed in native ecosystems, but the effect of inoculum build up within plantations on adjacent native eucalypt remnants is not known

Sensitivity towards DMI fungicides and haplotypic diversity of their CYP51 target in the Mycosphaerella graminicola population of Flanders

Septoria leaf blotch, caused by the fungus Mycosphaerella graminicola, is the most important wheat disease in Northwestern Europe, and is currently controlled by fungicide applications. Since the spread of resistance to methyl benzimidazole carbamates (MBCs) and quinone outside inhibitors (QoIs) in European countries, reliable control is mainly dependent upon sterol 14 alpha-demethylation inhibitors (DMIs). In the last decades however, a slow shift towards reduced sensitivity of M. graminicola to DMIs has been observed. This shift is caused mainly by mutations in the CYP51 gene encoding the 14 alpha-demethylase target protein for these fungicides. In this work, M. graminicola isolates were sampled at fields spread over Flanders, Belgium. In vitro assays were used to analyze the sensitivity of the Flemish M. graminicola population towards different DMIs. Sequencing of the CYP51 gene of these isolates allowed us to identify and map the haplotypes in this population. The results showed that there is a large variability in DMI sensitivity between the isolates, even within one field, which is reflected in a high diversity in CYP51 haplotypes within the M. graminicola population in Flanders. Next to some haplotypes that were not described in literature before, we found that the population is dominated by CYP51 haplotypes which were previously associated with increased resistance towards DMIs

Persistent, symptomless, systemic, and seed-borne infection of lettuce by Botrytis cinerea

Experiments are presented which show that Botrytis cinerea, the cause of gray mould disease, is often present in symptomless lettuce plants as a systemic, endophytic, infection which may arise from seed. The fungus was isolated on selective media from surface sterilized sections of roots, stem pieces and leaf discs from symptomless plants grown in a conventional glasshouse and in a spore-free air-flow provided by an isolation propagator. The presence of B. cinerea was confirmed by immuno-labelling the tissues with the Botrytis-specific monoclonal antibody BC-12.CA4. As plants grew, infection spread from the roots to stems and leaves. Surface sterilization of seeds reduced the number of infected symptomless plants. Artificial infection of seedlings with dry conidia increased the rate of infection in some experiments. Selected isolates were genetically finger-printed using microsatellite loci. This confirmed systemic spread of the inoculating isolates but showed that other isolates were also present and that single plants hosted multiple isolates. This shows that B. cinerea commonly grows in lettuce plants as an endophyte, as has already been shown for Primula. If true for other hosts, the endophytic phase may be as important a component of the species population as the aggressive necrotrophic phase