Improving on-farm surveillance strategies for tospoviruses and thrips to enhance the biosecurity of the nursery industry (NY19007)

Tospoviruses replicate within their insect vectors and persist through all larval and adult growth stages of these insects. These characteristics of vector transmission can be exploited for surveillance, as the thrips can be lured into traps using a combination of colours and pheromones, and the viruses directly detected within the thrips.  Pooled samples of thrips from the traps can then be interrogated using a combination of DNA barcoding and next generation sequencing. 

To pursue this activity, different universal PCR primers have been designed for both thrips and tospoviruses. These primers were selected based on sequence conservation at the protein level, which is indicative of biological constraints that restrict mutation and provides a strong basis for the universality of these primers. All primers have been tested on synthetic DNA templates, in order that the PCR assays can be properly optimized, the sensitivity of the diagnostic assays precisely quantified and the universality of the PCR primers empirically tested even if the target thrips species is exotic to Australia.  Finally, the universal PCR primers have been tested using thrips and tospovirus samples from Brisbane.   

The first group of primers target the COI gene of thrips, which is the standard gene for insect DNA barcoding. The most commonly cited primers in the literature are very old and designed before sequence data for thrips were available. Thus, there are many mismatches between these primers and thrips COI gene sequences. Researchers have relied upon using more complex thermocycling parameters to make the PCR work even with the primer mismatches. However, using such primers containing mismatches for metabarcoding most likely will lead to very biased species representation. To address this problem, complete COI gene sequences of thrips from complete mitochondrial genomes were aligned to identify the most conserved regions for designing universal primers. Three forward primers and three reverse primers that are universal to the thrips suborder Terebrantia have been designed, which includes the economically critical pests in the family Thripidae. The forward primers and the reverse primers can be used in different combinations to generate different sizes of amplicons, but all combinations include the region necessary for taxonomic assignment. Metabarcoding experiments will be conducted in the upcoming year for surveying thrips diversity in a wholesale nursery. 

The second group of PCR primers detect all known tospoviruses. More than 260 complete RdRp gene sequences of orthotospoviruses were analysed to identify the most conserved regions available for primer design. This primer set has been successfully used to amplify orthotospovirus sequences in both plant and thrips samples, as well as synthetic DNA templates representing viruses that are exotic to Australia.  

Finally, a primer pair targeting the EF1-alpha gene of insects has been designed using 880 insect sequences from the NCBI RefSeq database. The targeted gene region contains introns, and therefore different sized PCR amplicons are generated from genomic DNA and RNA. This PCR can therefore be used to assess RNA quality and therefore guard against false negative results for tospoviruses due to poor RNA quality.

The PhD candidate, Eyal Ziera, has completed an equivalent of one year and three months of his PhD. He successfully completed his confirmation of candidature on 4 October 2022 and is making excellent progress. The Research Progress Panel saw no impediments to the PhD program and approved Eyal’s continuation as a PhD candidate. He will undertake his next ad hoc review in April 2023. Additionally, Eyal presented some of his work at the Australasian Plant Virology Workshop in Melbourne in December 2023.

Eyal has made significant progress toward the end-to-end processes for metabarcoding which can be applied to pools of thrips caught by tapping flowers and on sticky traps. Eyal is currently testing sensitivity of metabarcoding for detecting one thrips in a pool of up to 1000 thrips. The metabacoding methodology will be submitted to a peer reviewed journal for publication in the next six months. During the establishment of the database of curated thrips species barcodes, three specimens belonging to the Frankliniella occidentalis ‘lupin’ haplotype were detected. This is the first detection of this haplotype in Australia.

During screening of plant samples for orthotospoviruses, additional isolates impatiens necrotic spot virus (INSV) were detected in Hoya from another Victorian nursery and also in Lisianthus from a flower nursery in NSW. The genomes of these additional INSV isolates will be sequenced and compared to the Victorian and NSW isolates that were collected previously. The information will contribute to the peer reviewed paper entitled ‘First Report of Impatiens necrotic spot orthotospovirus (INSV) in Australia’. This information will support further development of surveillance methodology for INSV and other orthotospoviruses.

Blue sticky traps have been set in two locations in Brisbane among beds of spider lilies (Hymenocallis spp.), which are very common hosts of tospoviruses, namely tomato spotted wilt virus (TSWV) and capsicum chlorosis virus (CaCV).

Thrips extracted from these traps have been identified by DNA barcoding and tested for the presence of tospoviruses using virus-specific assays.

The most frequently trapped thrips species are:

• Taeniothrips eucharii
• Thrips palmi
• Thrips tabaci
• Approximately 40 per cent of the T. eucharii specimens are viruliferous and carrying TSWV.

Project progress continues with...

• Preliminary in-field testing identified 18 plants, (including Gerbera, Osteospermum and tomato) from different sources infected with tomato spotted wilt orthotospovirus (TSWV) and five plants, (including sowthistle and Lisianthus), from different sources infected with impatiens necrotic spot orthotospovirus (INSV). Comparison of INSV isolates from lettuce in NSW and INSV isolates in lisianthius and sow thistle from VIC indicates that the NSW isolates are distinct from VIC isolates, suggesting two separate introductions to Australia.
• Further testing found TSWV in 14 different plant species and INSV in three different plant species. Planning for the development of technology for detecting orthotospoviruses is underway.
• Monitoring for orthotospoviruses in Victoria is ongoing with samples collected and tested at five different locations and across seasons in Victoria from a range of plant species, including commercial ornamental plants and weeds. Both TSWV and INSV was found in the samples with slightly higher number of TSWV recorded. Plans are underway to sequence and compare the full genome of TSWV and INSV isolates from different locations and host plants to reduce variability.
• An evaluation is underway to ensure the RT-PCR assays for testing Orthotospovirus species remain accurate and ensure no false negative or false positive results occur.
• More mechanical inoculation trials and transmission trials using thrips were performed with the purpose of infecting more plant species with INSV, however no successful transmission was recorded with the INSV inoculations. This work will continue as it will be important to assess the efficiency of INSV transmission by different vector species.
• The surveillance of thrips is progressing well with results showing that the non-destructive DNA extraction method used as part of this tool is inefficient for some thrips species, compared to destructive DNA extraction methods. Modifications to the method to ensure thrips remain intact for verification of presence by morphology are being evaluated.

Substantial progress has been made investigating the diversity of tospoviruses in Australia and developing molecular methods for thrips identification.

A potentially new tospovirus, called Pterostylis blotch virus (PtBV), was discovered more than 20 years ago but there has been insufficient evidence to assess whether it was an introduced or indigenous virus, and whether it poses a threat to Australian agriculture, including the Greenlife Industry. PtBV has now been fully characterised and there is compelling evidence that it likely originated in Australia and has had an ancient association with its native orchid host plants.

Research also began to investigate ornamental plant hosts of tospoviruses in Southeast Queensland. An accidental discovery from this research was the presence of the hibiscus strain of citrus leprosis virus C2 (CLV-C2H) in a hoya plant. This was its first ever detection in Australia, demonstrating that ornamental plant species have the potential to act as movement pathways for economically important plant pathogens, as CLV-C2H can cause a serious disease in citrus crops.

Onsite surveillance for thrips and orthotospoviruses was undertaken, with leaf samples collected every two to three months from major plants in the nursery motherstock in the glasshouse, trap plants located outside the nursery, and from various weed species. These samples will be tested for the presence of orthotospoviruses later this year. In addition, leaf samples from plants infected with tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) were collected from different cut flower nurseries in Victoria. The genomes of the Orthotospovirus isolates collected from various plants and sites will be compared to improve understanding of diversity, to aid infield diagnostic assay development.

As thrips are the vectors of orthotospoviruses, it is important to check the thrips species in different nurseries and seasons. Thrips were collected from different cut flower nurseries in Victoria, in which TSWV and INSV were detected. The thrips were identified morphologically and will be confirmed molecularly. Full analysis is underway and will be reported when complete.

The project team has partnered with the largest wholesale plant nursery in Queensland to find ways to enhance its pest surveillance program. They will deliver training in taxonomic skills to the nursery to improve their identification of the major thrips species, firstly using traditional morphological methods but later using simple molecular tests. Once experimental protocols are refined, the protocols will be extended more widely within the greenlife industry. 

Thrips are important nursery pests because they cause direct feeding damage to plants but also transmit tospoviruses. Consequently, a monitoring program for tospoviruses has begun, and several perennial ornamental plants identified as reservoirs of infection, namely spider lily, bridal bouquet and hoya.

The project team were surprised to discover both a tospovirus and citrus leprosis virus C2 (CLV-C2) in a hoya plant.  This represents the first report of CLV-C2 in Australia, and the presence of a potentially damaging pathogen for the citrus industry. This result demonstrates how economically important pathogens can be spread within nursery plants. Finally, a new tospovirus has been discovered in a native ground orchid, bringing the total number of species of tospovirus in Australia to five. This discovery needs to be considered in the design of a monitoring system for tospoviruses.