Brenda A. Coutts

Seed transmission of Wheat streak mosaic virus shown unequivocally in wheat

Under conditions that excluded any possibility of eriophyid mite vector activity, seed transmission of Wheat streak mosaic virus (WSMV) was shown in eight different wheat genotypes at rates of 0.5 to 1.5%. Virus identification in seedlings came from characteristic symptoms in wheat, enzyme-linked immunosorbent assay with WSMV-specific antibodies, reverse-transcription polymerase chain reaction tests with WSMV-specific primers, and cDNA sequence comparisons with published sequences. Sequence comparisons of four seedborne isolates showed ≥98.6% identity with the eight Australian isolates in GenBank, indicating a common seedborne origin of WSMV. These findings warrant reconsideration of currently accepted views on WSMV epidemiology and the likelihood of introducing it to new locations through planting untested wheat seed and the movement of germplasm.

Plant Virology and Next Generation Sequencing: Experiences with a Potyvirus

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.

The Epidemiology of Wheat streak mosaic virus in Australia: case histories, gradients, mite vectors, and alternative hosts

Wheat streak mosaic virus (WSMV) infection and infestation with its wheat curl mite (WCM; Aceria tosichella) vector were investigated in wheat crops at two sites in the low-rainfall zone of the central grainbelt of south-west Australia. In the 2006 outbreak, after a preceding wet summer and autumn, high WCM populations and total infection with WSMV throughout a wheat crop were associated with presence of abundant grasses and self-sown ‘volunteer’ wheat plants before sowing the field that became affected. Wind strength and direction had a major effect on WSMV spread by WCM to neighbouring wheat crops, the virus being carried much further downwind than upwind by westerly frontal winds. Following a dry summer and autumn in 2007, together with control of grasses and volunteer cereals before sowing and use of a different seed stock, no WSMV or WCM were found in the following wheat crop within the previously affected area or elsewhere on the same farm. In the 2007 outbreak, where the preceding summer and autumn were wet, a 40% WSMV incidence and WCM numbers that reached 4800 mites/ear at the margin of the wheat crop were associated with abundant grasses and volunteer wheat plants in adjacent pasture. WSMV incidence and WCM populations declined rapidly with increasing distance from the affected pasture. Also, wheat plants that germinated early had higher WSMV infection incidences than those that germinated later. The alternative WSMV hosts identified at these sites were volunteer wheat, annual ryegrass (Lolium rigidum), barley grass (Hordeum sp.), and wild oats (Avena fatua). In surveys outside the growing season at or near these two sites or elsewhere in the grainbelt, small burr grass (Tragus australianus), stink grass (Eragrostis cilianensis), and witch grass (Panicum capillare) were identified as additional alternative hosts.

Phylogenetic Analysis of Bean yellow mosaic virus Isolates from Four Continents : Relationship Between the Seven Groups Found and Their Hosts and Origins

Genetic diversity of Bean yellow mosaic virus (BYMV) was studied by comparing sequences from the coat protein (CP) and genome-linked viral protein (VPg) genes of isolates from four continents. CP sequences compared were those of 17 new isolates and 47 others already on the database, while the VPg sequences used were from four new isolates and 10 from the database. Phylogenetic analysis of the CP sequences revealed seven distinct groups, six polytypic and one monotypic. The largest and most genetically diverse polytypic group, which had intragroup diversity of 0.061 nucleotide substitutions per site, contained isolates from natural infections in eight host species. These original isolation hosts included both wild (four) and domesticated (four) species and were from monocotyledonous and dicotyledonous plant families, indicating a generalized natural host range strategy. Only one of the other five polytypic groups spanned both monocotyledons and dicotyledons, and all contained isolates from fewer species (one to four), all of which were domesticated and had lower intragroup diversity (0.019 to 0.045 nucleotide substitutions per site), indicating host specialization. Phylogenetic analysis of the fewer VPg sequences revealed three polytypic and two monotypic groupings. These groups also correlated with original natural isolation hosts, but the branch topologies were sometimes incongruous with those formed by CPs. Also, intragroup diversity was generally higher for VPgs than for CPs. A plausible explanation for the groups found when the 64 different CP sequences were compared is that the generalized group represents the original ancestral type from which the specialist host groups evolved in response to domestication of plants after the advent of agriculture. Data on the geographical origins of the isolates within each group did not reveal whether the specialized groups might have coevolved with their principal natural hosts where these were first domesticated, but this seems plausible.

Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences

Between 2006 and 2010, 5324 samples from at least 34 weed, two cultivated legume and 11 native species were collected from three cucurbit-growing areas in tropical or subtropical Western Australia. Two new alternative hosts of zucchini yellow mosaic virus (ZYMV) were identified, the Australian native cucurbit Cucumis maderaspatanus, and the naturalised legume species Rhyncosia minima. Low-level (0.7%) seed transmission of ZYMV was found in seedlings grown from seed collected from zucchini (Cucurbita pepo) fruit infected with isolate Cvn-1. Seed transmission was absent in >9500 pumpkin (C.maxima and C. moschata) seedlings from fruit infected with isolate Knx-1. Leaf samples from symptomatic cucurbit plants collected from fields in five cucurbit-growing areas in four Australian states were tested for the presence of ZYMV. When 42 complete coat protein (CP) nucleotide (nt) sequences from the new ZYMV isolates obtained were compared to those of 101 complete CP nt sequences from five other continents, phylogenetic analysis of the 143 ZYMV sequences revealed three distinct groups (A, B and C), with four subgroups in A (I-IV) and two in B (I-II). The new Australian sequences grouped according to collection location, fitting within A-I, A-II and B-II. The 16 new sequences from one isolated location in tropical northern Western Australia all grouped into subgroup B-II, which contained no other isolates. In contrast, the three sequences from the Northern Territory fitted into A-II with 94.6-99.0% nt identities with isolates from the United States, Iran, China and Japan. The 23 new sequences from the central west coast and two east coast locations all fitted into A-I, with 95.9-98.9% nt identities to sequences from Europe and Japan. These findings suggest that (i) there have been at least three separate ZYMV introductions into Australia and (ii) there are few changes to local isolate CP sequences following their establishment in remote growing areas. Isolates from A-I and B-II induced chlorotic symptoms in inoculated leaves of Chenopodium quinoa, but an isolate from A-II caused symptomless infection. One of three commercial ZYMV-specific antibodies did not detect all Australian isolates reliably by ELISA. A multiplex real-time PCR using dual-labelled probes was developed, which distinguished between Australian ZYMV isolates belonging to phylogenetic groups A-I, A-II and B-II.

Yield-limiting potential of necrotic and non-necrotic strains of bean yellow mosaic virus in narrow-leafed lupin (Lupinus angustifolius).

The yield losses caused by necrotic and non-necrotic strains of Bean yellow mosaic virus (BYMV) in narrow-leafed lupin (Lupinus angustifolius) were quantified in field experiments. Clover plants infected with either were introduced into plots to provide infection sources, and aphids spread infection to the lupin plants. When the effects of virus infection were examined in individual lupin plants infected with necrotic BYMV, they were killed by early infection so there was no seed production. With late infection, shoot dry wt, seed yield, and seed number were decreased by at least 55%, 80%, and 74%, respectively. With non-necrotic BYMV, shoot dry wt, seed yield, and seed number diminished with increasing duration of plant infection, these decreases ranging over 27-88%, 48-99%, and 35-98% for late to early infection, respectively. In partially infected stands in which both necrotic and non-necrotic BYMV were spreading, an additional incidence of 28% in plots with introduced non-necrotic strain foci over that in plots without introduced foci was sufficient to decrease overall seed yield significantly. However, an additional incidence of 10% was insufficient to do so in plots with introduced necrotic strain foci. In plots into which different numbers of clover plants infected with non-necrotic BYMV were introduced, subsequent incidence of infection depended on the magnitude of the initial virus source, and yield was decreased by 21-24%, 31-43%, and 64-66% with 4, 8, or 16 foci/plot, respectively. With both types of strain, yield loss in infected plants was mainly due to failure to produce any seed or to fewer seeds being produced, but smaller seed size also contributed. These results show that non-necrotic strains of BYMV have considerable yield-limiting potential in narrow-leafed lupin crops despite causing milder symptoms than necrotic strains. No evidence was obtained of seed-transmission of non-necrotic BYMV in narrow-leafed lupin, but a 0.2% seed transmission rate was detected in yellow lupin (Lupinus luteus).

Iris yellow spot virus found infecting onions in three Australian states

Iris yellow spot virus (IYSV) was detected for the first time in Australia, infecting onions in three and leeks in one state. Identification was confirmed using sap transmission to Nicotiana benthamiana, two IYSV-specific antisera in ELISA, RT-PCR with IYSV-specific primers, and sequence comparison with published IYSV sequences. Spring onion, onion seed and onion bulb crops were all infected, with spring onion being the most severely affected. The virus was also detected in nursery-grown onion and leek seedlings.

Indigenous and introduced potyviruses of legumes and Passiflora spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.

Localized Distribution of Iris yellow spot virus Within Leeks and Its Reliable Large-Scale Detection

In a survey to determine the incidence of Iris yellow spot virus (IYSV) in crops of several host species, samples of one leaf tip/plant were collected at random. When tested by enzyme-linked immunosorbent assay (ELISA) using IYSV-specific antibodies and a blocking step that improved test reliability, the virus was detected only in leek and onion. It was found in 11 of 21 leek and 2 of 26 onion plantings with apparent incidences of 1 to 7 and 1%, respectively. However, the figures for leek crops greatly underestimate IYSV incidence due to localization of infection within plants. Thus, in tests on multiple subsections from individual plants, IYSV was detected in one or more leaves but never in all leaves. Within infected leaves, it was localized in patches of infection found mainly in the middle and top subsections of the unfurled leaves, but infrequently in their bases. It never was found in the furled leaves that make up the stems, or in the basal plates or roots. Therefore, to obtain reliable estimates of IYSV incidences in largescale surveys of leek crops, the randomly collected samples tested by ELISA should consist of combined tissue subsections from the tops and middles of several leaves from each plant sampled.

Effects of Introduced and Indigenous Viruses on Native Plants: Exploring Their Disease Causing Potential at the Agro-Ecological Interface

The ever increasing movement of viruses around the world poses a major threat to plants growing in cultivated and natural ecosystems. Both generalist and specialist viruses move via trade in plants and plant products. Their potential to damage cultivated plants is well understood, but little attention has been given to the threat such viruses pose to plant biodiversity. To address this, we studied their impact, and that of indigenous viruses, on native plants from a global biodiversity hot spot in an isolated region where agriculture is very recent (<185 years), making it possible to distinguish between introduced and indigenous viruses readily. To establish their potential to cause severe or mild systemic symptoms in different native plant species, we used introduced generalist and specialist viruses, and indigenous viruses, to inoculate plants of 15 native species belonging to eight families. We also measured resulting losses in biomass and reproductive ability for some host–virus combinations. In addition, we sampled native plants growing over a wide area to increase knowledge of natural infection with introduced viruses. The results suggest that generalist introduced viruses and indigenous viruses from other hosts pose a greater potential threat than introduced specialist viruses to populations of native plants encountered for the first time. Some introduced generalist viruses infected plants in more families than others and so pose a greater potential threat to biodiversity. The indigenous viruses tested were often surprisingly virulent when they infected native plant species they were not adapted to. These results are relevant to managing virus disease in new encounter scenarios at the agro-ecological interface between managed and natural vegetation, and within other disturbed natural vegetation situations. They are also relevant for establishing conservation policies for endangered plant species and avoiding spread of damaging viruses to undisturbed natural vegetation beyond the agro-ecological interface.