Monica A. Kehoe

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.

Analyses of Twelve New Whole Genome Sequences of Cassava Brown Streak Viruses and Ugandan Cassava Brown Streak Viruses from East Africa: Diversity, Supercomputing and Evidence for Further Speciation

Cassava brown streak disease is caused by two devastating viruses, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) which are frequently found infecting cassava, one of sub-Saharan Africa’s most important staple food crops. Each year these viruses cause losses of up to

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

100 million USD and can leave entire families without their primary food source, for an entire year. Twelve new whole genomes, including seven of CBSV and five of UCBSV were uncovered in this research, doubling the genomic sequences available in the public domain for these viruses. These new sequences disprove the assumption that the viruses are limited by agro-ecological zones, show that current diagnostic primers are insufficient to provide confident diagnosis of these viruses and give rise to the possibility that there may be as many as four distinct species of virus. Utilizing NGS sequencing technologies and proper phylogenetic practices will rapidly increase the solution to sustainable cassava production.

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

Minimising losses caused by Zucchini yellow mosaic virus in vegetable cucurbit crops in tropical, sub-tropical and Mediterranean environments through cultural methods and host resistance

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.

Zucchini yellow mosaic virus: Contact Transmission, Stability on Surfaces, and Inactivation with Disinfectants

Between 2006 and 2009, 10 field experiments were done at Kununurra, Carnarvon or Medina in Western Australia (WA) which have tropical, sub-tropical and Mediterranean climates, respectively. These experiments investigated the effectiveness of cultural control measures in limiting ZYMV spread in pumpkin, and single-gene resistance in commercial cultivars of pumpkin, zucchini and cucumber. Melon aphids (Aphis gossypii) colonised field experiments at Kununurra; migrant green peach aphids (Myzus persicae) visited but did not colonise at Carnarvon and Medina. Cultural control measures that diminished ZYMV spread in pumpkin included manipulation of planting date to avoid exposing young plants to peak aphid vector populations, deploying tall non-host barriers (millet, Pennisetum glaucum) to protect against incoming aphid vectors and planting upwind of infection sources. Clustering of ZYMV-infected pumpkin plants was greater without a 25m wide non-host barrier between the infection source and the pumpkin plants than when one was present, and downwind compared with upwind of an infection source. Host resistance gene zym was effective against ZYMV isolate Knx-1 from Kununurra in five cultivars of cucumber. In zucchini, host resistance gene Zym delayed spread of infection (partial resistance) in 2 of 14 cultivars but otherwise did not diminish final ZYMV incidence. Zucchini cultivars carrying Zym often developed severe fruit symptoms (8/14), and only the two cultivars in which spread was delayed and one that was tolerant produced sufficiently high marketable yields to be recommended when ZYMV epidemics are anticipated. In three pumpkin cultivars with Zym, this gene was effective against isolate Cvn-1 from Carnarvon under low inoculum pressure, but not against isolate Knx-1 under high inoculum pressure, although symptoms were milder and marketable yields greater in them than in cultivars without Zym. These findings allowed additional cultural control recommendations to be added to the existing Integrated Disease Management strategy for ZYMV in vegetable cucurbits in WA, but necessitated modification of its recommendations over deployment of cultivars with resistance genes.

A proposal to help resolve the disagreement between naming of potato virus Y strain groups defined by resistance phenotypes and those defined by sequencing

In glasshouse experiments, Zucchini yellow mosaic virus (ZYMV) was transmitted from infected to healthy zucchini (Cucurbita pepo) plants by direct contact when leaves were rubbed against each other, crushed, or trampled, and, to a lesser extent, on ZYMV-contaminated blades. When sap from zucchini plants infected with three ZYMV isolates was kept at room temperature for up to 6 h, it infected healthy plants readily. Also, when sap from ZYMV-infected leaves was applied to seven surfaces (cotton, plastic, leather, metal, rubber vehicle tire, rubber-soled footwear, and human skin) and left for up to 48 h before the ZYMV-contaminated surface was rubbed onto healthy zucchini plants, ZYMV remained infective for 48 h on tire, 24 h on plastic and leather, and up to 6 h on cotton, metal, and footwear. On human skin, ZYMV remained infective for 5 min only. The effectiveness of 13 disinfectants at inactivating ZYMV was evaluated by adding them to sap from ZYMV-infected leaves which was then rubbed on to healthy zucchini plants. None of the plants became infected when nonfat dried milk (20%, wt/vol) or bleach (sodium hypochlorite at 42 g/liter, diluted 1:4) were used. When ZYMV-infected pumpkin leaves were trampled by footwear and then used to trample healthy plants, all plants became infected; however, when contaminated footwear was dipped in a footbath containing bleach (sodium hypochlorite at 42 g/liter, diluted 1:4) before trampling, none became infected. This study demonstrates that ZYMV can be transmitted by contact and highlights the need for on-farm hygiene practices (decontaminating tools, machinery, clothing, and so on) to be included in integrated disease management strategies for ZYMV in cucurbit crops.

Split Personality of a Potyvirus: To Specialize or Not to Specialize?

The complete coat protein (CP) nucleotide sequences of nine historical (1943-1984) potato virus Y (PVY) isolates belonging to resistance strain groupings YC, YZ or YO, and nine new Australian isolates from potato and tomato were compared with those of 85 others. New potato isolate BL was in resistance group YO. On phylogenetic analysis, the historical potato isolates fitted within phylogenetic groups C1 or C2 (YC), ‘YO’ (YZ, YO) or N-Wi (YZ), while the new isolates were in phylogenetic groups C1 (tomato) or ‘YO’ (potato, tomato). Substitution of the designation (YQ) for the current phylogenetic ‘YO’ grouping is proposed for consideration.

Hardenbergia mosaic virus: crossing the barrier between native and introduced plant species.

Bean yellow mosaic virus (BYMV), genus Potyvirus, has an extensive natural host range encompassing both dicots and monocots. Its phylogenetic groups were considered to consist of an ancestral generalist group and six specialist groups derived from this generalist group during plant domestication. Recombination was suggested to be playing a role in BYMVs evolution towards host specialization. However, in subsequent phylogenetic analysis of whole genomes, group names based on the original hosts of isolates within each of them were no longer supported. Also, nine groups were found and designated I-IX. Recombination analysis was conducted on the complete coding regions of 33 BYMV genomes and two genomes of the related Clover yellow vein virus (CYVV). This analysis found evidence for 12 firm recombination events within BYMV phylogenetic groups I–VI, but none within groups VII–IX or CYVV. The greatest numbers of recombination events within a sequence (two or three each) occurred in four groups, three which formerly constituted the single ancestral generalist group (I, II and IV), and group VI. The individual sequences in groups III and V had one event each. These findings with whole genomes are consistent with recombination being associated with expanding host ranges, and call into question the proposed role of recombination in the evolution of BYMV, where it was previously suggested to play a role in host specialization. Instead, they (i) indicate that recombination explains the very broad natural host ranges of the three BYMV groups which infect both monocots and dicots (I, II, IV), and (ii) suggest that the three groups with narrow natural host ranges (III, V, VI) which also showed recombination now have the potential to reduce host specificity and broaden their natural host ranges.

A proposal to rationalize within-species plant virus nomenclature: benefits and implications of inaction.

Hardenbergia mosaic virus (HarMV), genus Potyvirus, belongs to the bean common mosaic virus (BCMV) potyvirus lineage found only in Australia. The original host of HarMV, Hardenbergia comptoniana, family Fabaceae, is indigenous to the South-West Australian Floristic Region (SWAFR), where Lupinus spp. are grown as introduced grain legume crops, and exist as naturalised weeds. Two plants of H. comptoniana and one of Lupinus cosentinii, each with mosaic and leaf deformation symptoms, were sampled from a small patch of disturbed vegetation at an ancient ecosystem-recent agroecosystem interface. Potyvirus infection was detected in all three samples by ELISA and RT-PCR. After sequencing on an Illumina HiSeq 2000, three complete and two nearly complete HarMV genomes from H. comptoniana and one complete HarMV genome from L. cosentinii were obtained. Phylogenetic analysis which compared (i) the four new complete genomes with the three HarMV genomes on Genbank (two of which were identical), and (ii) coat protein (CP) genes from the six new genomes with the 38 HarMV CP sequences already on Genbank, revealed that three of the complete and one of the nearly complete new genomes were in HarMV clade I, one of the complete genomes in clade V and one nearly complete genome in clade VI. The complete HarMV genome from L. cosentinii differed by only eight nucleotides from one of the HarMV clade I genomes from a nearby H. comptoniana plant, with only one of these nucleotide changes being non-synonymous. Pairwise comparison between all the complete HarMV genomes revealed nucleotide identities ranging between 82.2% and 100%. Recombination analysis revealed evidence of two recombination events amongst the six complete genomes. This study provides the first report of HarMV naturally infecting L. cosentinii and the first example for the SWAFR of virus emergence from a native plant species to invade an introduced plant species.