Turnip yellows virus variants differ in host range, transmissibility, and virulence.

Turnip yellows virus (TuYV; family Solemoviridae, genus Polerovirus, species Turnip yellows virus) is a genetically diverse virus that infects a broad range of plant species across the world. Due to its global economic significance, most attention has been given to the impact of TuYV on canola (syn. oilseed rape; Brassica napus). In Australia, a major canola-exporting country, TuYV isolates are highly diverse, with the most variation concentrated in open reading frame 5 (ORF 5), which encodes the readthrough domain (P5) component of the readthrough protein (P3P5), which plays an important role in host adaptation and aphid transmission. When analysing ORF 5, Australian TuYV isolates form three phylogenetic groups with just 45 to 49% amino acid sequence identity: variants P5-I, P5-II, and P5-III. Despite the possible implications for TuYV epidemiology and management, research examining phenotypic differences between TuYV variants is scarce. This study was designed to test the hypothesis that three TuYV isolates, representing each of the Australian P5 variants, differ phenotypically. In particular, the host range, vector species, transmissibility, and virulence of isolates 5414 (P5-I5414), 5509 (P5-II5509), and 5594 (P5-III5594) were examined in a series of glasshouse experiments. Only P5-I5414 readily infected faba bean (Vicia faba), only P5-II5509 infected chickpea (Cicer arietinum), and only P5-I5414 and P5-III5594 infected lettuce (Lactuca sativa). Myzus persicae transmitted each isolate, but Brevicoryne brassicae and Lipaphis pseudobrassicae did not. When using individual M. persicae to inoculate canola seedlings, P5-I5414 had significantly higher transmission rates (82%) than P5-II5509 (62%) and P5-III5594 (59%). As indicated by enzyme-linked immunosorbent assay absorbance values, P5-I5414 reached higher virus titers in canola than P5-II5509, which, in turn, reached higher titers than P5-III5594. P5-I5414 was also more virulent in canola than P5-II5509 and P5-III5594, inducing more severe foliar symptoms, stunting, and, in one of two experiments, seed yield loss. Results from this study compared to those of previous studies suggest that analysis of ORF 5 alone is insufficient to assign isolates to coherent strain categories, and further sequencing and phenotyping of field isolates is required.

Vector species, pasture legume host range, and impact on grain legumes of an Australian soybean dwarf virus isolate.

Soybean dwarf virus (SbDV; family Tombusviridae, genus Luteovirus, species Soybean dwarf virus) can cause damaging disease epidemics in cultivated plants of the family Fabaceae. The biological characteristics of SbDV isolate WA-8, including its vector species, host range, and impact on Australian grain legume cultivars, were investigated in a series of glasshouse experiments. Isolate WA-8 was classified as the YP strain, as it was transmitted by Acyrthosiphon pisum (pea aphid) and Myzus persicae (green peach aphid) and infected known strain indicator species. Of the 18 pasture legume species inoculated with SbDV, 12 were SbDV hosts, including eight that had not been identified previously as hosts. When inoculated with SbDV, field pea (Pisum sativum), faba bean (Vicia faba), lentil (Lens culinaris), and narrow-leafed lupin cv. Jurien were the most susceptible (70 to 100% plant infection rates), and albus lupin (Lupinus albus), chickpea (Cicer arietinum), and narrow-leafed lupin cv. Mandelup were less susceptible (20 to 70%). Over the course of three experiments, chickpea was the most sensitive to infection, with a > 97% reduction in dry above-ground biomass (AGB) and a 100% reduction in seed yield. Field pea cv. Gunyah, faba bean, and lentil were also sensitive, with a 36 to 61% reduction in AGB. Field pea cv. Kaspa was relatively tolerant, with no significant reduction in AGB or seed yield. The information generated under glasshouse conditions in this study provides important clues for understanding SbDV epidemiology and suggests that it has the potential to cause damage to Australian grain legume crops in the field, especially if climate change facilitates its spread.

In-Field Capable Loop-Mediated Isothermal Amplification Detection of Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae Using a Rapid and Simple Crude Extraction Technique.

Fall armyworm, Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae), is an economically important pest worldwide and has recently been identified in Australia. Morphological identification of S. frugiperda at early larval stages can be difficult often requiring expert microscopy analysis. Rapid and accurate in-field diagnosis is vital for management decision support and there are no tools currently available for this purpose. In this study, a sensitive, specific, and in-field capable loop-mediated isothermal amplification (LAMP) assay was developed to detect S. frugiperda larvae. A primer set based on a highly conserved region of the S. frugiperda cytochrome oxidase subunit 1 (COX1) gene provided detection within 30 min from both total DNA and crude extractions. The crude extraction technique of crushing 10 mg of S. frugiperda material in 50 µl ddH2O and further diluting the homogenate in ddH2O is rapid, simple, and does not require heat blocks, centrifuges, or special buffers increasing its utility as a field-based technique. The primer set detected as little as 24 pg of S. frugiperda DNA and did not cross-react with any other of the lepidopteran species tested that are easily confused with S. frugiperda in Australia. Therefore, this assay could be used in-field to correctly identify the presence of S. frugiperda and thereby greatly assist with timely management decisions.

Impact of Turnip yellows virus infection on seed yield of an open-pollinated and hybrid canola cultivar when inoculated at different growth stages.

Turnip yellows virus (TuYV; family Luteoviridae, genus Polerovirus) is the most economically damaging virus infecting canola (Brassica napus) in the south-west Australian grainbelt. However, the impact of TuYV infection at different growth stages on canola seed yield has not been examined. This information is vital for implementing targeted management strategies. Four glasshouse experiments were conducted to examine seed yield losses incurred by an open-pollinated (ATR Bonito) and hybrid (Hyola® 404RR) canola cultivar when aphid-inoculated with TuYV at GS12 (two leaves unfolded), GS17 (seven leaves unfolded), GS30 (beginning of stem elongation) and GS65 (full flowering). When inoculated at GS12 and GS17, cv. Bonito plants incurred 30 % and 36 % seed yield losses, respectively, compared to healthy plants. Similarly, cv. 404RR incurred 41 % and 26 % seed yield losses at GS12 and GS17, respectively. However, when inoculated at GS30, whilst cv. Bonito plants incurred a 26 % seed yield loss, cv. 404RR incurred no significant loss. Neither cultivar incurred seed yield losses from inoculation at GS65. Additional information was collected from these experiments to improve sampling protocols to enhance TuYV detection, with a molecular and serological technique. When canola plants were at pre-flowering growth stages, TuYV was reliably detected 7-14 days after inoculation (DAI) in the youngest leaf. Once flowering had begun, TuYV was consistently detected 7-14 DAI in petals and flower buds. In contrast, regardless of growth stage, testing the oldest leaf regularly resulted in delayed detection or false negatives. Information generated in this study helps to quantify the value of management strategies targeted at preventing TuYV spread in pre-flowering canola crops and ultimately increase the efficiency of resource use.

In-field capable loop-mediated isothermal amplification detection of Turnip yellows virus in plants and its principal aphid vector Myzus persicae.

Widespread Turnip yellows virus (TuYV) infection causes severe seed yield and quality losses in rapeseed (Brassica napus) crops grown in broadacre agricultural systems worldwide. Current TuYV detection protocols are expensive and time consuming, and can have poor specificity and sensitivity. Typically, they are used as a diagnostic tool to test already symptomatic plants, limiting their practical value to reactive disease management. To improve diagnostic services so that they provide earlier, cheaper, faster, more specific and sensitive TuYV detection, novel and innovative protocols that utilise new technology are required. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect TuYV in crude and total RNA extractions of leaf material and its principal aphid vector Myzus persicae. The assay was based on a set of six primers, highly sensitive and specific to TuYV, derived from a TuYV isolate originating from the south-west Australian grainbelt. TuYV was readily detected in 1 in 100 dilutions of (i) infected to uninfected leaf material, and (ii) viruliferous to non-viruliferous M. persicae. Furthermore, detection was successful in a majority of aphids stored for at least 8 weeks in various trapping and storage substances, including 30% ethylene glycol, sticky trap glue and 70% ethanol. This RT-LAMP assay protocol enables quicker and cheaper diagnosis for TuYV than currently adopted laboratory-based diagnostic techniques. Ultimately, it has the potential for earlier in-field TuYV detection in combination with aphid trapping surveillance programs.

Forecasting model for Pea seed-borne mosaic virus epidemics in field pea crops in a Mediterranean-type environment.

An empirical model was developed to forecast Pea seed-borne mosaic virus (PSbMV) incidence at a critical phase of the annual growing season to predict yield loss in field pea crops sown under Mediterranean-type conditions. The model uses pre-growing season rainfall to calculate an index of aphid abundance in early-August which, in combination with PSbMV infection level in seed sown, is used to forecast virus crop incidence. Using predicted PSbMV crop incidence in early-August and day of sowing, PSbMV transmission from harvested seed was also predicted, albeit less accurately. The model was developed so it provides forecasts before sowing to allow sufficient time to implement control recommendations, such as having representative seed samples tested for PSbMV transmission rate to seedlings, obtaining seed with minimal PSbMV infection or of a PSbMV-resistant cultivar, and implementation of cultural management strategies. The model provides a disease forecast risk indication, taking into account predicted percentage yield loss to PSbMV infection and economic factors involved in field pea production. This disease risk forecast delivers location-specific recommendations regarding PSbMV management to end-users. These recommendations will be delivered directly to end-users via SMS alerts with links to web support that provide information on PSbMV management options. This modelling and decision support system approach would likely be suitable for use in other world regions where field pea is grown in similar Mediterranean-type environments.

Establishing alighting preferences and species transmission differences for Pea seed-borne mosaic virus aphid vectors.

Pea seed-borne mosaic virus (PSbMV) infection causes a serious disease of field pea (Pisum sativum) crops worldwide. The PSbMV transmission efficiencies of five aphid species previously found landing in south-west Australian pea crops in which PSbMV was spreading were studied. With plants of susceptible pea cv. Kaspa, the transmission efficiencies of Aphis craccivora, Myzus persicae, Acyrthosiphon kondoi and Rhopalosiphum padi were 27%, 26%, 6% and 3%, respectively. Lipaphis erysimi did not transmit PSbMV in these experiments. The transmission efficiencies found for M. persicae and A. craccivora resembled earlier findings, but PSbMV vector transmission efficiency data were unavailable for A. kondoi, R. padi and L. erysimi. With plants of partially PSbMV resistant pea cv. PBA Twilight, transmission efficiencies of M. persicae, A. craccivora and R. padi were 16%, 12% and 1%, respectively, reflecting putative partial resistance to aphid inoculation. To examine aphid alighting preferences over time, free-choice assays were conducted with two aphid species representing efficient (M. persicae) and inefficient (R. padi) vector species. For this, alatae were set free on multiple occasions (10-15 repetitions each) amongst PSbMV-infected and mock-inoculated pea or faba bean (Vicia faba) plants. Following release, non-viruliferous R. padi alatae exhibited a general preference for PSbMV-infected pea and faba bean plants after 30min-4h, but preferred mock-inoculated plants after 24h. In contrast, non-viruliferous M. persicae alatae alighted on mock-inoculated pea plants preferentially for up to 48h following their release. With faba bean, M. persicae preferred infected plants at the front of assay cages, but mock-inoculated ones their backs, apparently due to increased levels of natural light there. When preliminary analyses were performed to detect PSbMV-induced changes in the volatile organic compound profiles of pea and faba bean plants, higher numbers of volatiles representing a range of compound groups (such as aldehydes, ketones and esters) were found in the headspaces of PSbMV-infected than of mock-inoculated pea or faba bean plants. This indicates PSbMV induces physiological changes in these hosts which manifest as altered volatile emissions. These alterations could be responsible for the differences in alighting preferences. Information from this study enhances understanding of virus-vector relationships in the PSbMV-pea and faba bean pathosystems.

Pea seed-borne mosaic virus Pathosystem Drivers under Mediterranean-Type Climatic Conditions: Deductions from 23 Epidemic Scenarios.

Drivers of Pea seed-borne mosaic virus (PSbMV) epidemics in rainfed field pea crops were examined under autumn to spring growing conditions in a Mediterranean-type environment. To collect aphid occurrence and PSbMV epidemic data under a diverse range of conditions, 23 field pea data collection blocks were set up over a 6-year period (2010 to 2015) at five locations in the southwest Australian grain-growing region. PSbMV infection levels in seed sown (0.1 to 13%), time of sowing (22 May to 22 June), and cultivar (Kaspa or PBA Twilight) varied with location and year. Throughout each growing season, rainfall data were collected, leaf and seed samples were tested to monitor PSbMV incidence in the crop and transmission from harvested seed, and sticky traps were used to monitor flying aphid numbers. Winged migrant Acyrthosiphon kondoi, Lipaphis erysimi, Myzus persicae, and Rhopalosiphum padi were identified in green tile traps in 2014 and 2015. However, no aphid colonization of field pea plants ever occurred in the blocks. The deductions made from collection block data illustrated how the magnitude of PSbMV spread prior to flowering is determined by two primary epidemic drivers: (i) PSbMV infection incidence in the seed sown, which defines the magnitude of virus inoculum source for within-crop spread by aphids, and (ii) presowing rainfall that promotes background vegetation growth which, in turn, drives early-season aphid populations and the time of first arrival of their winged migrants to field pea crops. Likely secondary epidemic drivers included wind-mediated PSbMV plant-to-plant contact transmission and time of sowing. PSbMV incidence at flowering time strongly influenced transmission rate from harvested seed to seedlings. The data collected are well suited for development and validation of a forecasting model that informs a Decision Support System for PSbMV control in field pea crops.

Pea seed-borne mosaic virus: Stability and Wind-Mediated Contact Transmission in Field Pea.

Pea seed-borne mosaic virus (PSbMV) stability in sap and its contact transmission between field pea plants were investigated in glasshouse experiments. When infective leaf sap was kept at room temperature and inoculated to plants in the absence of abrasive, it was still highly infective after 6 h and low levels of infectivity remained after 30 h. PSbMV was transmitted from infected to healthy plants by direct contact when leaves were rubbed against each other. It was also transmitted when intertwining healthy and PSbMV-infected plants were blown by a fan to simulate wind. When air was blown on plants kept at 14 to 20°C, contact transmission of PSbMV occurred consistently and the extent of transmission was enhanced when plants were dusted with diatomaceous earth prior to blowing. In contrast, when plants were kept at 20 to 30°C, blowing rarely resulted in transmission. No passive contact transmission occurred when healthy and infected plants were allowed to intertwine together. This study demonstrates that PSbMV has the potential to be transmitted by contact when wind-mediated wounding occurs in the field. This may play an important role in the epidemiology of the virus in field pea crops, especially in situations where contact transmission expands initial crop infection foci before aphid arrival.

Pea seed-borne mosaic virus in Field Pea: Widespread Infection, Genetic Diversity, and Resistance Gene Effectiveness.

From 2013 to 2015, incidences of Pea seed-borne mosaic virus (PSbMV) infection were determined in semi-leafless field pea (Pisum sativum) crops and trial plots growing in the Mediterranean-type environment of southwest Australia. PSbMV was found at incidences of 2 to 51% in 9 of 13 crops, 1 to 100% in 20 of 24 cultivar plots, and 1 to 57% in 14 of 21 breeding line plots. Crops and plots of 'PBA Gunyah', 'Kaspa', and 'PBA Twilight' were frequently PSbMV infected but none of PSbMV resistance gene sbm1-carrying 'PBA Wharton' plants were infected. In 2015, 14 new PSbMV isolates obtained from these various sources were sequenced and their partial coat protein (CP) nucleotide sequences analyzed. Sequence identities and phylogenetic comparison with 39 other PSbMV partial CP nucleotide sequences from GenBank demonstrated that at least three PSbMV introductions have occurred to the region, one of which was previously unknown. When plants of 'Greenfeast' and PBA Gunyah pea (which both carry resistance gene sbm2) and PBA Wharton and 'Yarrum' (which carry sbm1) were inoculated with PSbMV pathotype P-2 isolate W1, resistance was overcome in a small proportion of plants of each cultivar, showing that resistance-breaking variants were likely to be present. An improved management effort by pea breeders, advisors, and growers is required to diminish infection of seed stocks, avoid sbm gene resistance being overcome in the field, and mitigate the impact of PSbMV on seed yield and quality. A similar management effort is likely to be needed in field pea production elsewhere in the world.