Epidemiology of Zucchini yellow mosaic virus in cucurbit crops in a remote tropical environment

Highlights

• Critical knowledge of tropical Zucchini yellow mosaic virus (ZYMV) epidemics revealed.

• Comprehensive Integrated Management strategy for tropical ZYMV epidemics devised.

• ZYMV spread within data collection blocks resembled spread in commercial cucurbit crops.

• Aphid vector numbers caught varied widely with trap location and time of year.

• No evidence of new ZYMV variants introduced since 1996 obtained.

Abstract

In the remote Ord River Irrigation Area (ORIA) in tropical northwest Australia, severe Zucchini yellow mosaic virus (ZYMV) epidemics threaten dry season (April-October) cucurbit crops. In 2016–2017, wet season (November-March) sampling studies found a low incidence ZYMV infection in wild Cucumis melo and Citrullus lanatus var. citroides plants, and both volunteer and garden crop cucurbits. Such infections enable its persistence in the wet season, and act as reservoirs for its spread to commercial cucurbit crops during the dry season. Tests on 1019 samples belonging to 55 species from 23 non-cucurbitaceous plant families failed to detect ZYMV. It was also absent from wild cucurbit weeds within sandalwood plantations. The transmission efficiencies of a local isolate by five aphid species found in the ORIA were: 10 % (Aphis craccivora), 7% (A. gossypii), 4% (A. nerii), and 0% (Rhopalosiphum maidis and Hysteroneura setariae). In 2016–2017, in all-year-round trapping at five representative sites, numbers of winged aphids caught were greatest in July-August (i.e. mid growing season) but varied widely between trap sites reflecting local aphid host abundance and year. Apart from one localised exception in 2017, flying aphid numbers caught and ZYMV spread in data collection blocks during 2015–2017 resembled what occurred commercial cucurbit crops. When ZYMV spread from external infection sources into melon blocks, its predominant spread pattern consisted of 1 or 2 plant infection foci often occurring at their margins. In addition, when plants of 29 cucurbit cultivars were inoculated with an ORIA isolate and two other ZYMV isolates and the phenotypes elicited were compared, they resembled each other in overall virulence. However, depending upon isolate-cultivar combination, differences in symptom expression and severity occurred, and one isolate caused a systemic hypersensitive phenotype in honeydew melon cvs Estilo and Whitehaven. When the new genomic RNA sequences of 19 Australian isolates were analysed, all seven ORIA isolates fitted within ZYMV phylogroup B, which also included two from southwest Australia, whereas the remaining 10 isolates were all within minor phylogroups A–I or A–II. Based on previous research and the additional knowledge of ZYMV epidemic drivers established here, an integrated disease management strategy targeting ZYMV spread was devised for the ORIA’s cucurbit industry.

Introduction

Zucchini yellow mosaic virus (ZYMV; family Potyviridae, genus Potyvirus) causes a damaging disease of cucurbit crops, especially in the world’s tropical and subtropical regions (Desbiez and Lecoq, 1997; Coutts et al., 2011a; Lecoq and Katis, 2014; CABI, 2016; Keinath et al., 2017). It has 680–730 nm flexuous filamentous virions (Lisa et al., 1981; Desbiez and Lecoq, 1997, and a positive sense, single stranded RNA genome consisting of 9951 nucleotides (nts) (Lin et al., 2001). Several aphid species transmit the virus non-persistently (Castle et al., 1992; Yuan and Ullman, 1996; Desbiez and Lecoq, 1997; Katis et al., 2006), and it is also contact transmissible via wounds (Coutts et al., 2013). Seed transmission of ZYMV occurs occasionally in squash and zucchini (Cucurbita pepo), and in one pumpkin species (Cucurbita maxima) (Davis and Mizuki, 1986; Greber et al., 1987, 1988; Schrijnwerkers et al., 1991; Robinson et al., 1993; Fletcher et al., 2000; Riedle-Bauer et al., 2002; Tobias and Palkovics, 2003; Tobias et al., 2008; Coutts et al., 2011b), but is not reported from melon (Cucumis melo), cucumber (Cucumis sativus), watermelon (Citrullus lanatus) or a second species of cultivated pumpkin (Cucurbita moschata) (Provvidenti and Robinson, 1987; Greber et al., 1987, 1988; Gleason and Provvidenti, 1990; Robinson et al., 1993; Desbiez and Lecoq, 1997; Riedle-Bauer et al., 2002; Glasa and Kollerova, 2007; Coutts et al., 2011c). Obvious foliage symptoms develop in ZYMV-infected cucurbit plants and infected fruits are sometimes rendered unmarketable due to malformation and surface discolouration. Early ZYMV infection causes severe fruit yield losses and up to 100 % yield reduction of marketable fruit (Blua and Perring, 1989; Desbiez and Lecoq, 1997; Fletcher et al., 2000; Coutts et al., 2011a).

In addition to spreading ZYMV from infected curcurbit crops or volunteer cucurbit plants, aphid vectors acquire it from nearby infected alternative hosts and spread it to healthy cucurbit crops growing nearby (Desbiez and Lecoq, 1997; Lecoq and Desbiez, 2008; Lecoq and Katis, 2014). However, although its epidemics occur commonly in cucurbit crops in many world regions, naturally occurring infected alternative hosts have proven difficult to find as their occurrence is sporadic and their incidence very low (Desbiez and Lecoq, 1997; Lecoq and Katis, 2014; Lecoq et al., 2014; Coutts et al., 2011a,b). Worldwide, recorded alternative ZYMV hosts include: volunteer cucurbits, wild cucurbits (Al-Musa, 1989; Delmiglio and Pearson, 2006; Perring et al., 1992; Coutts et al., 2011b), non-cucurbitaceous weed species from several families (Al-Musa, 1989; Desbiez and Lecoq, 1997; Perring et al., 1992; Riedle-Bauer et al., 2002; Svoboda and Polak, 2002), and several ornamental plant species (Choi et al., 2002; Chen and Hong, 2008). In addition, aphid vectors spread ZYMV within cucurbit crops by acquiring it from infected seedlings growing within newly planted crops derived from sowing infected seed stocks or from transplanting seedlings obtained from ZYMV-contaminated nurseries (Lecoq and Desbiez, 2008; Lecoq and Katis, 2014). Nearby discarded infected fruits also act as virus sources for aphid vectors to acquire ZYMV and spread it to crops (Lecoq et al., 2003). Moreover, transporting infected cucurbit seed stocks, seedlings and fruits to new locations all constitute means by which ZYMV, or damaging new strains of it, get introduced to cucurbit crops growing regions previously free of them (Desbiez and Lecoq, 1997; Lecoq and Desbiez, 2008; Lecoq and Katis, 2014). Another way it can be transported over large distances, including over oceans, is through viruliferous aphid vectors flying from distant ZYMV-infected crops being transported considerable distances by wind currents subsequently landing in healthy cucurbit crops (Maina et al., 2017a, 2019).

In Australia, cucurbit crops are widely grown for domestic consumption and export markets. In the horticultural cropping regions of tropical and sub-tropical Australia, disease caused by ZYMV greatly diminishes yield and quality of produce, and cucurbit industry profitability. The vegetable cucurbits squash, zucchini, cucumber and pumpkin, and fruit cucurbits melon and watermelon are all severely affected (Greber et al., 1987, 1988; Büchen-Osmond et al., 1988; Coutts and Jones, 2005; Coutts et al., 2011a). ZYMV epidemics cause severe losses in cucurbit crops growing in the Kimberley region of northern Western Australia (WA) and in the Northern Territory (NT), both of which have tropical climates, and in the subtropical Gascoyne region of WA (Jones, 1996; Coutts and Jones, 2005; Coutts et al., 2011a). They also cause severe losses in cucurbit crops in tropical and subtropical Queensland (QLD), and subtropical Northern New South Wales (Greber et al., 1987, 1988; Büchen-Osmond et al., 1988; Herrington et al., 1988a,b). In Australia, Aphis gossypii (melon aphid) and M. persicae (green peach aphid) are recorded as being ZYMV vectors (Greber et al., 1988; Coutts et al., 2011a), and ZYMV seed transmission has been found in squash and zucchini, but not in watermelon, melon or pumpkin (Greber et al., 1987, 1988; Coutts et al., 2011b). Alternative hosts found ZYMV-infected at low incidence are volunteer watermelon (Citrullus lanatus var. lanatus), wild Afghan melon (Citrullus lanatus var. citroides), Cucumis myriocarpus (prickly paddy melon), Cucumis maderaspatanus (ivy gourd) (all Cucurbitaceae) and the legume weed Rhynchosia minima (snout bean) (Büchen-Osmond et al., 1988; McLean and Price, 1984; Coutts and Jones, 2005; Coutts et al., 2011b). Phylogenetic analysis of coat protein (CP) genes (Coutts et al., 2011b) and complete genomes (Maina et al., 2017a, 2019) of Australian ZYMV isolates, revealed their presence in three distinct ZYMV lineages. Isolates in two of them were restricted to remote cucurbit growing regions in the continents’ tropical north, one in Ord River Irrigation Area (ORIA) in the East Kimberley (major phylogroup B; the tropical ‘Southeast Asian grouping’), and the other in Darwin and Katherine in the NT (minor phylogroup A–II). The third grouping was present elsewhere in the continent (minor phylogroup A–I).

The ORIA’s creation in 1963 followed clearing of native bushland, completion of a Diversion Dam across the Ord River and construction of the Kununurra township to service the irrigation scheme. In 1967, the main Ord River Dam was completed upstream of the Diversion Dam creating Lake Argyle, the biggest man made lake in Australia, making 5600 gigalitres of water available for irrigation https://www.lakeargyle.com/history-statistics-environment/ord-river-irrigation-scheme. Flood irrigation enables the ORIA’s rich fertile soils to grow a diverse range of annual and perennial crops. However, currently 50 % of its area is occupied by Indian sandalwood (Santalum album) plantations that produce oil for the perfume industry. Melons, watermelons and pumpkins are widely grown commercially in the cooler dry season (April-October). In 1996, a survey of ORIA cucurbit crops found infection with ZYMV, Papaya ringspot virus (PRSV; family Potyviridae, genus Potyvirus) and Squash mosaic virus (SqMV; family Secoviridae, genus Comovirus) (Jones, 1996; Maina et al., 2017b). In 2003–2004, ORIA surveys found ZYMV, PRSV, SqMV and Watermelon mosaic virus (WMV; family Potyviridae, genus Potyvirus). ZYMV was the commonest virus infecting 74 % of crops often at high incidences, while WMV was least common (Coutts and Jones, 2005). All ZYMV isolates sequenced from the ORIA belonged to phylogroup B (Coutts et al., 2011b; Maina et al., 2017a). In 2006 and 2009, field experiments with pumpkin investigated the effectiveness of three cultural control measures (manipulation of planting date, non-host barriers, planting upwind) and single-gene resistance at diminishing ZYMV spread, and other field experiments studied its temporal and spatial dynamics of spread (Coutts et al., 2011a).The findings from these experiments were used to adjust the ZYMV Integrated Disease Management (IDM) strategy for WA of Coutts and Jones (2005) to make it more effective (Coutts et al., 2011a). However, although helpful in reducing virus incidence, this adjustment did not prevent further annual ZYMV epidemics and losses from occurring in the ORIA, especially in late sown cucurbit crops.

Epidemiological understanding of the ORIA’s tropical ZYMV-cucurbit pathosystem is limited to knowledge that volunteer watermelon and R. minima can act as alternative ZYMV hosts (Coutts and Jones, 2005; Coutts et al., 2011b), information on its spatiotemporal dynamics of spread from marginal or internal ZYMV sources within pumpkin crops (Coutts et al., 2011c), and records that eight aphid species are present. The eight aphid species reported are Aphis craccivora (cowpea aphid), A. gossypii (melon aphid), A. nerii (oleander aphid), Hysteroneura setariae (rusty plum aphid), Rhopalosiphum maidis (corn aphid), R. rufiabdominalis (rice root aphid, syn. Tetraneura nigriabdominalis), Schoutedenia ralumensis (bougainvillea aphid) (http://www.planthealthaustralia.com.au/resources/australian-plant-pest-database), and R. padi (Coutts et al., 2011a). Of these, only A. craccivora, A. gossypii, R. maidis and R. padi are known ZYMV vectors, the latter two species only transmitting at very low efficiencies (Castle et al., 1992; Yuan and Ullman, 1996; Greber et al., 1988; Garzo et al., 2004; Katis et al., 2006). No information is available for the ORIA on: (i) which aphid species are important vectors of local ZYMV isolates; (ii) which aphid hosts occur other than cucurbit volunteer and crop plants; (iii) how both ZYMV and aphids persist when commercial cucurbit crops are absent during the annual wet season (November to March), and (iv) which climatic and other factors drive aphid population build-up and ZYMV epidemic development in commercial cucurbit crops during the dry season (April to October). This paper addresses (i) and (iv), and the ZYMV component of (iii). Our research also addressed (ii) and the aphid component of (iii), but our findings on these aspects were reported separately (Clarke et al., 2020, accompanying paper). In brief, Clarke et al. (2020) found aphids all-year-round; overall, 38 % of leaf samples of introduced weed, Australian native plant, and volunteer or planted crop plant species collected from diverse ORIA sites were aphid-colonised; and amongst the 23 plant families sampled 19 families contained aphid-colonised species. Of the six aphid species found, A. nerii, H. setariae, R. maidis and S. ralumensis each colonised 1–3 plant species from a single plant family, A. craccivora colonised 14 species in five families and A. gossypii colonised 19 species in 11 families. Understory weeds and host trees in sandalwood plantations were important reservoirs for spread of A. craccivora to wild and crop plant hosts. Alternative hosts growing in rural bushland, irrigation channel banks, vacant or fallow land, and orchard plantation understories also constituted important aphid vector reservoirs.

The aims of our research were to discover which hosts ZYMV persists in, and where they occur, during the annual ORIA wet season, and which climatic and other factors drive aphid population build-up and ZYMV epidemic development in commercial cucurbit crops during the dry season. We used the resulting improved understanding of ZYMV epidemics in the ORIA, along with knowledge from previous research, to devise an IDM strategy specifically targeting ZYMV epidemics in ORIA cucurbit crops. Also, to investigate whether any new ZYMV variants have become established in the ORIA or elsewhere in Australia, we undertook phylogenetic analysis of the complete genomes of seven additional ORIA and 12 other isolates from around the Australian continent. In addition, to test the belief that ZYMV isolates from the ORIA induce more severe disease symptoms on cucurbits than other Australian isolates, we compared the symptoms induced when an ORIA and two other ZYMV isolates were inoculated to plants of 29 cucurbit cultivars.