Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes and pathogenesis-related genes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h postinoculation with S. sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S. sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S. sclerotiorum.

Developing effective fumigation protocols to manage strongly phosphine-resistant Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae)

BACKGROUND The emergence of high levels of resistance in Cryptolestes ferrugineus (Stephens) in recent years threatens the sustainability of phosphine, a key fumigant used worldwide to disinfest stored grain. We aimed at developing robust fumigation protocols that could be used in a range of practical situations to control this resistant pest. RESULTS Values of the lethal time to kill 99.9% (LT99.9, in days) of mixed-age populations, containing all life stages, of a susceptible and a strongly resistant C. ferrugineus population were established at three phosphine concentrations (1.0, 1.5 and 2.0 mg L−1) and three temperatures (25, 30 and 35 °C). Multiple linear regression analysis revealed that phosphine concentration and temperature both contributed significantly to the LT99.9 of a population (P < 0.003, R2 = 0.92), with concentration being the dominant variable, accounting for 75.9% of the variation. Across all concentrations, LT99.9 of the strongly resistant C. ferrugineus population was longest at the lowest temperature and shortest at the highest temperature. For example, 1.0 mg L−1 of phosphine is required for 20, 15 and 15 days, 1.5 mg L−1 for 12, 11 and 9 days and 2.0 mg L−1 for 10, 7 and 6 days at 25, 30 and 35 °C, respectively, to achieve 99.9% mortality of the strongly resistant C. ferrugineus population. We also observed that phosphine concentration is inversely proportional to fumigation period in regard to the population extinction of this pest. CONCLUSION The fumigation protocols developed in this study will be used in recommending changes to the currently registered rates of phosphine in Australia towards management of strongly resistant C. ferrugineus populations, and can be repeated in any country where this type of resistance appears. © 2014 Commonwealth of Australia. Pest Management Science © 2014 Society of Chemical Industry

Susceptibility to sulfuryl fluoride and lack of cross-resistance to phosphine in developmental stages of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

BACKGROUND Our aim was to ascertain the potential of sulfuryl fluoride (SF) as an alternative fumigant to manage phosphine-resistant pests. We tested the susceptibility of all life stages of red flour beetle, Tribolium castaneum (Herbst), to SF and assessed the presence of cross-resistance to this fumigant in phosphine-resistant strains of this species. RESULTS Analysis of dose–response data indicated that the egg was the stage most tolerant to SF under a 48 h exposure period. At LC50, eggs were 29 times more tolerant than other immature stages and adults, and required a relatively high concentration of 48.2 mg L−1 for complete mortality. No significant differences in tolerance to SF were observed among the three larval instars, pupae and adults, and all of these stages were controlled at a low concentration of 1.32 mg L−1. Phosphine-resistant strains did not show cross-resistance to SF. CONCLUSION Our research concluded that the current maximum registered rate of SF, 1500 gh m−3, is adequate to control all the post-embryonic life stages of T. castaneum over a 48 h fumigation period, but it will fail to achieve complete mortality of eggs, indicating the risk of some survival of eggs under this short exposure period. As there is no cross-resistance to SF in phosphine-resistant insects, it will play a key role in managing phosphine resistance in stored-grain insect pests. © 2014 Commonwealth of Australia. Pest Management Science © 2014 Society of Chemical Industry

Native Phloem and Wood Borers in Australian Mediterranean Forest Trees

Native Mediterranean forests in Australia are dominated by two tree genera, Eucalyptus and Acacia, while Pinus and Eucalyptus dominate plantation forestry. In native forests, there is a high diversity of phloem and wood borers across several families in the Coleoptera and Lepidoptera. In the Coleoptera, cerambycid beetles (Cerambycidae), jewel beetles (Buprestidae), bark, ambrosia and pinhole beetles (Curculionidae) and pinworms (Lymexelidae) are some of the most commonly found beetles attacking eucalypts and acacias. In the Lepidoptera, wood moths (Cossidae), ghost moths (Hepialidae) and borers in the Xyloryctidae (subfamily Xyloryctinae) are most common. In contrast to native forests, there is a much more limited range of native insects present in Australian plantations, particularly in exotic Pinus spp. plantations, although eucalypt plantations do share some borers in common with native forests. This chapter reviews the importance of these borers in Australian forests primarily from an economic perspective (i.e. those species that cause damage to commercial tree species) and highlights a paucity of native forest species that commonly kill trees relative to the large scales regularly seen in North America and Europe.

Impact of the invasive rust Puccinia psidii (myrtle rust) on native Myrtaceae in natural ecosystems in Australia

The invasive rust Puccinia psidii (myrtle rust) was detected in Australia in 2010 and is now established along the east coast from southern New South Wales to far north Queensland. Prior to reaching Australia, severe damage from P. psidii was mainly restricted to exotic eucalypt plantations in South America, guava plantations in Brazil, allspice plantations in Jamaica, and exotic Myrtaceous tree species in the USA; the only previous record of widespread damage in native environments is of endangered Eugenia koolauensis in Hawai’i. Using two rainforest tree species as indicators of the impact of P. psidii, we report for the first time severe damage to endemic Myrtaceae in native forests in Australia, after only 4 years’ exposure to P. psidii. A 3-year disease exclusion trial in a natural stand of Rhodamnia rubescens unequivocally showed that repeated, severe infection leads to gradual crown loss and ultimately tree mortality; trees were killed in less than 4 years. Significant (p < 0.001) correlations were found between both incidence (r = 0.36) and severity (r = 0.38) of P. psidii and subsequent crown loss (crown transparency). This provided supporting evidence to conclude a causal association between P. psidii and crown loss and tree mortality in our field assessments of R. rubescens and Rhodomyrtus psidioides across their native range. Assessments revealed high levels of damage by P. psidii to immature leaves, shoots and tree crowns—averaging 76 % (R. rubescens) and 95 % (R. psidioides) crown transparency—as well as tree mortality. For R. psidioides, we saw exceptionally high levels of tree mortality, with over half the trees surveyed dead and 40 % of stands with greater than 50 % tree mortality, including two stands where all trees were dead. Tree mortality was less prevalent for R. rubescens, with only 12 % of trees surveyed dead and two sites with greater than 50 % mortality. Any alternative causal agents for this tree mortality have been discounted. The ecological implications of this are unclear, but our work clearly illustrates the potential for P. psidii to negatively affect Australia’s biodiversity.

Sorption and desorption of sulfuryl fluoride by wheat, flour and semolina

Sulfuryl fluoride (SF) has been developed as a fumigant for control of insect pests in stored grain. However, there is very limited information on the sorption behaviour of this fumigant, which can be critical to its bioactivity, application and potential for residues. We undertook a comprehensive laboratory study of the sorption and desorption of SF by wheat (bread and durum), flour and semolina at 15, 25 and 35 °C, moisture contents 12% and 15%, and concentration × time combinations at CT = 1500 mgh/L (4.167 mg/L × 360 h, 8.928 mg/L × 168 h and 31.25 mg/L × 48 h). At each dosage, sorption rate increased as commodity temperature and moisture content increased. The highest rates of sorption occurred at 35 °C and 15% m.c., and lowest rates at 15 °C and 12% m.c., and the rate was independent of initial concentration. Sorption followed first order reaction kinetics described by the exponential decay equation, Ct = C0·e−k*t, where k is the sorption rate constant. The most important factors determining the rate of sorption were commodity particle size (exposed surfaces) and temperature. Little sorption of fumigant occurred within the first 24 h whereas longer fumigation times resulted in significant sorption. Unbound SF was rapidly lost from the commodity upon aeration with no further desorption detected under any of the test conditions.

Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes

Phosphine (PH3) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.

Genetic conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.)

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam and Australia. Each of four strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from two independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mgL-1 for a 48 hour exposure at 25°C. Thus, the mechanism of high level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Responses of fruit flies (Tephritidae: Dacinae) to novel male attractants in north Queensland, Australia, and improved lures for some pest species

Male fruit fly attractants, cue-lure and methyl eugenol (ME), have been successfully used for the last 50 years in the monitoring and control of Dacini fruit flies (Bactrocera and Dacus species). However, over 50% of Dacini are non-responsive to either lure, including some pest species. A new lure, zingerone, has been found to weakly attract cue- and ME-responsive species in Malaysia. In Australia it attracted a weakly cue-responsive minor pest Bactrocera jarvisi (Tryon) and three non-responsive' species. Similar compounds were tested in Queensland and attracted cue- and ME-responsive species and two non-responsive' species. In this study, 14 novel compounds, including raspberry ketone formate (RKF) (Melolure) and zingerone, were field tested in comparison with cue-lure and ME at 17 sites in north Queensland. The most attractive novel lures were isoeugenol, methyl-isoeugenol, dihydroeugenol and zingerone. Several non-responsive' species responded to the new lures: Bactrocera halfordiae (Tryon), a species of some market access concern, was most attracted to isoeugenol; B.barringtoniae (Tryon), B.bidentata (May) and B.murrayi (Perkins) responded to isoeugenol, methyl-isoeugenol and dihydroeugenol; two new species of Dacus responded to zingerone. Bactrocera kraussi (Hardy), a cue-responsive minor pest in north Queensland, was significantly more attracted to isoeugenol than cue-lure. The cue-responsive D.absonifacies (May) and D.secamoneaeDrew were significantly more attracted to zingerone than cue-lure. Bactrocera yorkensisDrew & Hancock, a ME-responsive species was significantly more attracted to isoeugenol, methyl-isoeugenol and dihydroeugenol than ME. The preferential response to RKF or cue-lure was species specific. Six species were significantly more attracted to RKF, including the pests B.tryoni (Froggatt), B.frauenfeldi (Schiner) and minor pest B.bryoniae (Tryon); eight species were significantly more attracted to cue-lure including the pest B.neohumeralis (Hardy). These findings have significance in the search for optimal male lures for pest species elsewhere in the world.

Strategies to limit the impact of nematode pressure on sugarcane productivity in the Isis

Two trials were done in this project. One was a continuation of work started under a previous GRDC/SRDC-funded activity, 'Strategies to improve the integration of legumes into cane based farming systems'. This trial aimed to assess the impact of trash and tillage management options and nematicide application on nematodes and crop performance. Methods and results are contained in the following publication: Halpin NV, Stirling GR, Rehbein WE, Quinn B, Jakins A, Ginns SP. The impact of trash and tillage management options and nematicide application on crop performance and plant-parasitic nematode populations in a sugarcane/peanut farming system. Proc. Aust. Soc. Sugar Cane Technol. 37, 192-203. Nematicide application in the plant crop significantly reduced total numbers of plant parasitic nematodes (PPN) but there was no impact on yield. Application of nematicide to the ratoon crop significantly reduced sugar yield. The study confirmed other work demonstrating that implementation of strategies like reduced tillage reduced populations of total PPN, suggesting that the soil was more suppressive to PPN in those treatments. The second trial, a variety trial, demonstrated the limited value of nematicide application in sugarcane farming systems. This study has highlighted that growers shouldn’t view nematicides as a ‘cure all’ for paddocks that have historically had high PPN numbers. Nematicides have high mammalian toxicity, have the potential to contaminate ground water (Kookana et al. 1995) and are costly. The cost of nematicide used in R1 was approx. $320 - $350/ha, adding $3.50/t of cane in a 100 t/ha crop. Also, our study demonstrated that a single nematicide treatment at the application rate registered for sugarcane is not very effective in reducing populations of nematode pests. There appears to be some levels of resistance to nematodes within the current suite of varieties available to the southern canelands. For example the soil in plots that were growing Q183 had 560% more root knot nematodes / 200mL soil compared to plots that grew Q245. The authors see great value in investment into a nematode screening program that could rate varieties into groups of susceptibility to both major sugarcane nematode pests. Such a rating could then be built into a decision support ‘tree’ or tool to better enable producers to select varieties on a paddock by paddock basis.

Two Complete Genome Sequences of Phasey Bean Mild Yellows Virus, a Novel Member of the Luteoviridae from Australia

We present here the complete genome sequences of a novel polerovirus from Trifolium subterraneum (subterranean clover) and Cicer arietinum (chickpea) and compare these to a partial viral genome sequence obtained from Macroptilium lathyroides (phasey bean). We propose the name phasey bean mild yellows virus for this novel polerovirus.