Screening of Diverse Lupinus spp. Highlights New Resistances to Sclerotinia sclerotiorum.

Stem rot caused by Sclerotinia sclerotiorum is a serious, and sometimes devastating, disease of lupin (Lupinus spp.). Two hundred and thirty-six lupin accessions from across 12 Lupinus species were screened against the prevalent S. sclerotiorum isolate MBRS-1 (pathotype 76). L. angustifolius accession 21655 and L. albus var. albus accession 20589 showed immune and 'near-immune' responses, respectively. Thirteen accessions of L. angustifolius, three accessions each of L. albus and L. albus var. albus, and a single accession each of L. albus var. graecus, L. mutabilis, L. palaestinus and L. pilosus (totalling ~4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, two accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totalling ~10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, one L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, WW-3 (pathotype 10). Against this isolate, five L. angustifolius accessions showed a HR response and four showed a R response, and the L. digitatus accession showed a moderate resistance (MR) response. Overall, isolate WW-3 caused significantly (P<0.05) smaller lesions than MBRS-1 across tested accessions in common. In addition, 328 plants in a 'wild' naturalized field population of L. cosentini were screened in situ in the field against isolate MBRS-1. Five (~1.5%) of the 328 plants of wild lupin showed an immune response, 63 (~19%) showed a HR response, and 146 (~45%) showed a R response. We believe this is the first examination of diverse Lupinus spp. germplasm responses to a prevalent pathotype of S. sclerotiorum. Lupin genotypes exhibiting high level resistance to Sclerotinia stem rot identified in this study can now be used as parental lines for crosses in lupin breeding programs and/or directly as improved cultivars to reduce the adverse impact of this disease on lupin crops.

Genetic and molecular analysis of stem rot (Sclerotinia sclerotiorum) resistance in Brassica napus (canola type).

Identifying the molecular and genetic basis of resistance to Sclerotinia stem rot (Sclerotinia sclerotiorum) is critical for developing long-term and cost-effective management of this disease in rapeseed/canola (Brassica napus). Current cultural or chemical management options provide, at best, only partial and/or sporadic control. Towards this, a B. napus breeding population (Mystic x Rainbow), including the parents, F1, F2, BC1P1 and BC1P2, was utilized in a field study to determine the inheritance pattern of Sclerotinia stem rot resistance (based on stem lesion length, SLL). Broad sense heritability was 0.58 for SLL and 0.44 for days to flowering (DTF). There was a significant negative correlation between SLL and stem diameter (SD) (r = -0.39) and between SLL and DTF (r = -0.28), suggesting co-selection of SD and DTF traits, along with SLL, should assist in improving overall resistance. Non-additive genetic variance was evident for SLL, DTF, and SD. In a genome wide association study (GWAS), a significant quantitative trait locus (QTL) was identified for SLL. Several putative candidate marker trait associations (MTA) were located within this QTL region. Overall, this study has provided valuable new understanding of inheritance of resistance to S. sclerotiorum, and has identified QTL, MTAs and transgressive segregants with high-level resistances. Together, these will foster more rapid selection for multiple traits associated with Sclerotinia stem rot resistance, by enabling breeders to make critical choices towards selecting/developing cultivars with enhanced resistance to this devastating pathogen.

Novel Resistances Provide New Avenues to Manage Alternaria Leaf Spot (Alternaria brassicae) in Canola (Brassica napus), Mustard (B. juncea), and Other Brassicaceae Crops.

Alternaria leaf spot (Alternaria brassicae) can be a devastating disease in canola (Brassica napus) and mustard (B. juncea), but there are no highly effective host resistances available. Screening of 150 diverse Brassicaceae varieties under glasshouse conditions highlighted important novel resistances. In particular, Camelina sativa '4076' and Diplotaxis erucoides 'Wasabi Rocket' had complete resistance across disease assessment parameters (leaf incidence [%LDI]; severity [%LAD]; consequent defoliation [%LCI]). The next most resistant varieties were C. sativa 'CSA' (%LDI 0.6; %LAD 0.4), '4144' (%LDI 1.2; %LAD 0.5), '405' (%LDI 1.7; %LAD 0.7), C. sativa '3274' (%LDI 2.5; %LAD 0.8), Carrichtera annua 'CAN3' (%LDI 7.7; %LAD 4.0), and Sisymbrium irio 'London Rocket' (%LDI 2.1; %LAD 0.8), all with %LCI values of 0. Other genotypes showing high-level resistance included S. erysimoides 'SER 4' (%LDI 11.8; %LAD 5.6; %LCI 0) and D. cardaminoides 'Wild Rocket' (%LDI 15.5; %LAD 7.2; %LCI 0), and those showing moderate resistance were Brassica carinata 'ML-EM-1' (Rungwe), B. insularis 'Moris', B. napus 'ZY006', B. oxyrrhina 'BOX1', B. oleracea var. capitata 'Sugarloaf', B. tournefortii 'CN01-104-2', and Sinapis alba 'Concerta' with %LDI 21.6 to 29.8, %LAD 12.8 to 21.0, and %LCI 0 to 5.7. In particular, B. napus 'ZY006' for canola and B. oleracea var. capitata 'Sugarloaf' can now be directly utilized (i.e., without crossing impairment) for Brassica species and vegetable breeding programs, respectively. While all B. juncea genotypes were susceptible, there were some less susceptible varieties from India in comparison with genotypes from Australia or China. The most susceptible test genotype was Rapistrum sativus (%LDI 89.4; %LAD 83.9; %LCI 71.0), highlighting the value of the resistances identified. These findings not only highlight a range of novel resistances against A. brassicae for canola, mustard, and other diverse Brassicaceae breeding programs to develop resistant commercial varieties, but also emphasize highly susceptible varieties to avoid in both breeding programs and commercial situations conducive to Alternaria leaf spot.

Faba Bean Gall Pathogen Physoderma viciae: New Primers Reveal Its Puzzling Association with the Field Pea Ascochyta Complex.

Recent morphological and molecular studies confirmed Physoderma viciae, and not Olpidium viciae, to be the causative agent of the devastating Faba Bean Gall (FBG) disease on faba bean (Vicia faba) in Ethiopia and also highlighted its ability to cross-infect with other host genera such as Pisum and Trifolium. In this study, the first pair of specific primer 'Physo 1' and primer pair 'Physo D' are reported from molecular sequences of this pathogen from the conserved LSU (S28) gene. Whereas 'Physo 1' readily detects P. viciae, 'Physo D', clearly separates its identity from the common and confounding presence of Didymella/Phoma spp. The study also reports the presence of the Ascochyta blight pathogen complex, symptomless but almost universal on field pea (Pisum sativum), within faba bean infested by P. viciae. We emphasize historical evidence confirming such unique association in other legumes, such as the subterranean clover (Trifolium subterraneum). This new finding has significant implications for rotations involving different legume crop and/or forage legume genera and possibly provides the first explanation for the widespread occurrence of the field pea Ascochyta blight pathogen complex even in the absence of field pea cropping for many years.

Quantitative Inheritance of Sclerotinia Stem Rot Resistance in Brassica napus and Relationship to Cotyledon and Leaf Resistances.

Sclerotinia sclerotiorum is a necrotrophic fungus causing devastating stem rot and associated yield losses of canola/rapeseed (Brassica napus) worldwide, including in Australia. Developing host resistance against Sclerotinia stem rot is critical if this disease in canola/rapeseed is to be successfully managed, as cultural or chemical control options provide only partial or sporadic control. Three B. napus breeding populations, C2, C5 and C6, including the parents, F1, F2, BC1P1, and BC2P2, were used in a field study with an objective of exploring the inheritance pattern of disease resistance (based on stem lesion length [SLL]) and the genetic relationships of disease with stem diameter (SD) or days to first flowering (DTF), and to compare these new adult plant stem resistances against S. sclerotiorum with those of seedling (cotyledon and leaf) resistances in earlier studies. Heritability (broad sense) of SLL was 0.57 and 0.73 for population C2 at 3 and 5 weeks postinoculation and 0.21 for population C5 at 5 weeks postinoculation. Additive genetic variance was evident within all 3 populations for DTF but not for SD. Narrow-sense heritability for DTF was 0.48 (C2), 0.42 (C5), and 0.32 (C6). SD, DTF, and SLL were all inherited independently, with no significant genetic covariance between traits in bivariate analysis. Genetic variance for SLL in populations C2 and C5 was entirely nonadditive, and there was significant nonadditive genetic covariance of SLL at 3 and 5 weeks postinoculation. Generation means analysis in population C2 supported the conclusion that complex epistatic interactions controlled SLL. Several C2 and C5 progeny showed high adult plant stem resistance, which may be critical in developing enhanced stem resistance in canola/rapeseed. Although population C6 showed no genetic variation for SLL resistance in this study, it showed significant nonadditive genetic variance at the cotyledon and leaf stages in earlier studies. We conclude that host resistance varies across different plant growth stages, and breeding must be targeted for resistance at each growth stage. In populations C2, C5, and C6, resistance to S. sclerotiorum in stem, leaf, and cotyledon was always controlled by nonadditive effects such as complex epistasis or dominance. Overall, our findings in relation to the quantitative inheritance of Sclerotinia stem rot resistance, together with the new high-level resistances identified, will enable breeders to select/develop genotypes with enhanced resistances to S. sclerotiorum.

Pathogen Biocontrol Using Plant Growth-Promoting Bacteria (PGPR): Role of Bacterial Diversity.

A vast microbial community inhabits in the rhizosphere, among which, specialized bacteria known as Plant Growth-Promoting Rhizobacteria (PGPR) confer benefits to host plants including growth promotion and disease suppression. PGPR taxa vary in the ways whereby they curtail the negative effects of invading plant pathogens. However, a cumulative or synergistic effect does not always ensue when a bacterial consortium is used. In this review, we reassess the disease-suppressive mechanisms of PGPR and present explanations and illustrations for functional diversity and/or stability among PGPR taxa regarding these mechanisms. We also provide evidence of benefits when PGPR mixtures, rather than individuals, are used for protecting crops from various diseases, and underscore the critical determinant factors for successful use of PGPR mixtures. Then, we evaluate the challenges of and limitations to achieving the desired outcomes from strain/species-rich bacterial assemblages, particularly in relation to their role for plant disease management. In addition, towards locating additive or synergistic outcomes, we highlight why and how the benefits conferred need to be categorized and quantified when different strains/species of PGPR are used in combinations. Finally, we highlight the critical approaches needed for developing PGPR mixtures with improved efficacy and stability as biocontrols for utilization in agricultural fields.

Comparative analysis of draft genome assemblies developed from whole genome sequences of two Hyaloperonospora brassicae isolate samples differing in field virulence on Brassica napus.

Hyaloperonospora brassicae causes downy mildew, a major disease of Brassicaceae species. We sequenced the genomes of two H. brassicae isolates of high (Sample B) and low (Sample C) virulence. Sequencing reads were first assembled de novo with software's SOAPdenovo2, ABySS V2.1 and Velvet V1.1 and later combined to create meta-assemblies with genome sizes of 72.762 and 76.950Mb and predicted gene densities of 1628 and 1644 /Mb, respectively. We could annotate 12.255 and 13,030 genes with high proportions (91-92%) of complete BUSCOs for Sample B and C, respectively. Comparative analysis revealed conserved and varied molecular machinery underlying the physiological specialisation and infection capabilities. BLAST analysis against PHI gene database suggested a relatively higher loss of genes for virulence and pathogenicity in Sample C compared to Sample B, reflecting pathogen evolution through differential rates of mutation and selection. These studies will enable identification and monitoring of H. brassicae virulence factors prevailing in-field.

Dimorphism in Neopseudocercosporella capsellae, an Emerging Pathogen Causing White Leaf Spot Disease of Brassicas.

White leaf spot pathogen: Neopseudocercosporella capsellae causes significant damage to many economically important Brassicaceae crops, including oilseed rape through foliar, stem, and pod lesions under cool and wet conditions. A lack of information on critical aspects of the pathogen's life cycle limits the development of effective control measures. The presence of single-celled spores along with multi-celled conidia on cotyledons inoculated with multi-celled conidia suggested that the multi-celled conidia were able to form single-celled spores on the host surface. This study was designed to demonstrate N. capsellae morphological plasticity, which allows the shift between a yeast-like single-celled phase and the multi-celled hyphal phase. Separate experiments were designed to illustrate the pathogen's morphological transformation to single-celled yeast phase from multi-celled hyphae or multi-celled macroconidia in-vitro and in-planta. Results confirmed the ability of N. capsellae to switch between two morphologies (septate hyphae and single-celled yeast phase) on a range of artificial culture media (in-vitro) or in-planta on the host surface before infection occurs. The hyphae-to-yeast transformation occurred through the production of two morphologically distinguishable blastospore (blastoconidia) types (meso-blastospores and micro-blastospores), and arthrospores (arthroconidia).

Phoma medicaginis Isolate Differences Determine Disease Severity and Phytoestrogen Production in Annual Medicago spp.

Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield but can reduce herbage quality by inducing production of phytoestrogens (particularly coumestrol and 4'-O-methylcoumestrol), which can also reduce the ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (Medicago truncatula cultivar Cyprus, Medicago polymorpha var. brevispina cultivar Serena, and Medicago murex cultivar Zodiac) after inoculation with 35 isolates of P. medicaginis. Across the isolate × cultivar combinations, leaf disease incidence, petiole/stem disease incidence, leaf disease severity, petiole disease severity, and leaf yellowing severity ranged up to 100, 89.4, 100, 58.1, and 61.2%, respectively. Cultivars Cyprus and Serena were the most susceptible and cultivar Zodiac was the most resistant to P. medicaginis. Isolates WAC3653, WAC3658, and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1,995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars and across the three cultivars overall, where r = 0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P < 0.05). Although cultivar Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cultivar Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cultivar Serena but only a 7-fold increase in cultivar Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.

Challenges With Managing Disease Complexes During Application of Different Measures Against Foliar Diseases of Field Pea.

Studies were undertaken across five field locations in Western Australia to determine the relative changes in disease severity and subsequent field pea yield from up to four foliar pathogens associated with a field pea foliar disease complex (viz. genera Didymella, Phoma, Peronospora, and Septoria) across four different pea varieties sown at three different times and at three different densities. Delaying sowing of field pea significantly (P < 0.05) reduced the severity of Ascochyta blight (all five locations) and Septoria blight (one location), increased the severity of downy mildew (four locations), but had no effect on seed yield. In relation to Ascochyta blight severity at 80 days after sowing, at all locations the early time of sowing had significantly (P < 0.05) more severe Ascochyta blight than the mid and late times of sowing. Increasing actual plant density from 20 to 25 plants m-2 to 58 to 78 plants m-2 significantly (P < 0.05) increased the severity of the Ascochyta blight (four locations) and downy mildew (one location), and it increased seed yield at four locations irrespective of sowing date and three locations irrespective of variety. Compared with varieties Dundale, Wirrega, and Pennant, variety Alma showed significantly (P < 0.05) less severe Ascochyta blight, downy mildew, and Septoria blight (one location each). Grain yield was highest for the early time of sowing at three locations. Varieties Alma, Dundale, and Wirrega significantly (P < 0.05) outyielded Pennant at four locations. The percentage of isolations of individual Ascochyta blight pathogens at 80 days after the first time of sowing varied greatly, with genus Didymella ranging from 25 to 93% and genus Phoma ranging from 6 to 23% across the five field locations. This fluctuating nature of individual pathogen types and proportions within the Ascochyta blight complex, along with variation in the occurrence of pathogens Peronospora and Septoria, highlights the challenges to understand and manage the complexities of co-occurring different foliar pathogens of field pea. While the search for more effective host resistance continues, there is a need for and opportunities from further exploring and exploiting cultural management approaches focusing on crop sequence diversification, intercropping, manipulating time of sowing and stand density, and application of improved seed sanitation and residue/inoculum management practices. We discuss the constraints and opportunities toward overcoming the challenges associated with managing foliar disease complexes in field pea.

Novel Disease Host Resistances in the World Core Collection of Trifolium subterraneum.

Glasshouse and field investigations of the phenotypic expressions of resistance of a 97-member World Core Collection of subterranean clover (Trifolium subterraneum) collected from its native Mediterranean habitat and representing approximately 80% of the total genetic diversity within the known 10,000 accessions of the species against the most important damping-off and root rot (Phytophthora clandestina, and Pythium irregulare) and foliar (Kabatiella caulivora, Uromyces trifolii-repentis, and Erysiphe trifoliorum) pathogens were performed. An additional 28 diverse cultivars were also included. Associations of these genotypes among 18 disease parameters and 17 morphological traits, and among these disease parameters and 24 climatic and eco-geographic variables from their collection sites, were examined. Many genotypes showed strong phenotypic expression of novel host disease resistance against one or more pathogens, enabling their potential deployment as disease-resistant parents in subterranean clover breeding programs. These new sources of resistance enable future "pyramiding" of different resistance genes to improve resistance against these pathogens. Of particular value were genotypes with multiple disease-resistance across soilborne and/or foliar diseases, because many of these pathogens co-occur. All diseases had some parameters significantly correlated with one or more morphological traits and with one or more sites of origin variables. In particular, there were significant negative correlations between damping-off (i.e., germination) and 8 of the 17 morphological characters. The outcomes of these studies provide crucial information to subterranean clover breeding programs, enabling them to simultaneously select genotypes with multiple resistance to co-occurring soilborne and foliar diseases and desirable traits to offer renewed hope for re-establishing a more productive subterranean clover livestock feedbase despite multiple diseases prevailing widely.

Evidence for Niche Differentiation in the Environmental Responses of Co-occurring Mucoromycotinian Fine Root Endophytes and Glomeromycotinian Arbuscular Mycorrhizal Fungi.

Fine root endophytes (FRE) were traditionally considered a morphotype of arbuscular mycorrhizal fungi (AMF), but recent genetic studies demonstrate that FRE belong within the subphylum Mucoromycotina, rather than in the subphylum Glomeromycotina with the AMF. These findings prompt enquiry into the fundamental ecology of FRE and AMF. We sampled FRE and AMF in roots of Trifolium subterraneum from 58 sites across temperate southern Australia. We investigated the environmental drivers of composition, richness, and root colonization of FRE and AMF by using structural equation modelling and canonical correspondence analyses. Root colonization by FRE increased with increasing temperature and rainfall but decreased with increasing phosphorus (P). Root colonization by AMF increased with increasing soil organic carbon but decreased with increasing P. Richness of FRE decreased with increasing temperature and soil pH. Richness of AMF increased with increasing temperature and rainfall but decreased with increasing soil aluminium (Al) and pH. Aluminium, soil pH, and rainfall were, in decreasing order, the strongest drivers of community composition of FRE; they were also important drivers of community composition of AMF, along with temperature, in decreasing order: rainfall, Al, temperature, and soil pH. Thus, FRE and AMF showed the same responses to some (e.g. soil P, soil pH) and different responses to other (e.g. temperature) key environmental factors. Overall, our data are evidence for niche differentiation among these co-occurring mycorrhizal associates.

Canola Growth Stage at Time of Infection Determines Magnitude of White Leaf Spot (Neopseudocercosporella capsellae) Impact.

White leaf spot (Neopseudocercosporella capsellae) is a persistent and increasingly important foliar disease for canola (Brassica napus) across southern Australia. To define the role of plant growth stage in the development of disease epidemics, we first investigated the response of different canola cultivars (Scoop and Charlton) at five Sylvester-Bradley growth stages against N. capsellae. White leaf spot disease incidence and severity was dependent on plant growth stage and cultivar (both P < 0.001), with plants being most susceptible at plant growth stage 1.00 (cotyledon stage) followed by plant growth stage 1.04 (fourth leaf stage). Then, to quantify the impact of this disease on canola yield, we investigated the in-field relationship of white leaf spot disease incidence and severity with seed yield loss following artificial inoculation commencing at growth stage 1.04 (fourth leaf stage). White leaf spot significantly (P < 0.001) reduced seed yield by 24% in N. capsellae inoculated field plots compared with noninoculated field plots. To our knowledge, this is the first time that serious seed yield losses from this disease have been quantified in the field. The current study demonstrates that N. capsellae disease incidence and severity on canola is determined by host growth stage at which pathogen infestation occurs. Emerging seedling cotyledons were highly susceptible, followed by less susceptibility in first true leaves to emerge, but then increasing susceptibility as plants subsequently aged toward the fourth leaf stage. This explains field observances where white leaf spot readily establishes on emerging seedlings and subsequently becomes more prevalent and severe as plants age.

White Leaf Spot Caused by Neopseudocercosporella capsellae: A Re-emerging Disease of Brassicaceae.

White leaf spot can cause significant damage to many economically important Brassicaceae crops, including oilseed rape, vegetable, condiment, and fodder Brassica species, and recently has been identified as a re-emerging disease. The causal agent, Neopseudocercosporella capsellae, produces foliar, stem, and pod lesions under favorable weather conditions. N. capsellae secretes cercosporin, a non-host specific, photo-activated toxin, into the host tissue during the early infection process. The pathogen has an active parasitic stage on the living host and a sexual or asexual saprobic stage on the dead host. Where the sexual stage exists, ascospores initiate the new disease cycle, while in the absence of the sexual stage, conidia produced by the asexual stage initiate new disease cycles. Distribution of the pathogen is worldwide; however, epidemiology and disease severity differ between countries or continents, with it being more destructive in Subtropical, Mediterranean, or Temperate climate regions with cool and wet climates. The pathogen has a wide host range within Brassicaceae. Brassica germplasm show varied responses from highly susceptible to completely resistant to pathogen invasion and significant susceptibility differences are observed among major crop species. Cultural practices only provide effective disease control when the climate is not conducive. An increase in the susceptible host population and favorable weather conditions have together favored the recent rise in white leaf spot disease occurrence and spread. The lack of understanding of variation in pathogen virulence and associated resistant gene sources within brassicas critically limits the potential to develop efficient control measures.

Temperature Drives Contrasting Alternaria Leaf Spot Epidemic Development in Canola and Mustard Rape from Alternaria japonica and A. brassicae.

Recent surveys of canola (Brassica napus) crops across southern Australia highlighted that Alternaria leaf spot on canola is not solely caused by Alternaria brassicae but that other Alternaria spp. are also involved, including A. japonica. Studies were undertaken into the effects of different temperatures (14 and 10°C [day and night] or 22 and 17°C [day and night]) on development of Alternaria leaf spot caused by A. japonica as compared with A. brassicae in cotyledons (embryonic leaves) and true leaves (first leaves) of canola (B. napus 'Thunder TT') and mustard rape (B. juncea 'Dune'). Both pathogens expressed less disease at lower temperatures of 14 and 10°C with percent disease index (%DI) of 19.1 for A. japonica and 41.8 for A. brassicae, but expressed significantly more disease at higher temperatures of 22 and 17°C with %DI of 80.8 and 88.2 for the same pathogens, respectively. At 14 and 10°C, mustard rape cotyledons showed less disease (percent cotyledons disease index [%CDI] = 18.1) from A. japonica but showed more disease (%CDI = 75.0) from A. brassicae. However, at 22 and 17°C, cotyledons and true leaves of both canola and mustard rape showed significantly more disease and varied in expressing the disease severity to the two pathogens; true leaves of mustard rape showed less disease (percent true leaf disease index [%TDI] = 48.4) from A. japonica but showed more disease (%TDI = 92.0) from A. brassicae. At 22 and 17°C, cotyledons of canola expressed more disease from A. japonica (%CDI = 99.1) than from A. brassicae (%CDI = 70.7). At the lower temperature, both host species showed the least disease, with mean %DI of 27.3 and 33.5 for canola and mustard rape, respectively, as compared with the higher temperatures, where there was a greater DI, with %DI values of 87.9 and 81.2 for these same host species, respectively. We believe that these are the first studies to highlight the critical role played by temperature for A. japonica as compared with A. brassicae in Alternaria leaf spot disease development and severity. These findings explain how temperature affects Alternaria leaf spot severity caused by A. japonica as compared with A. brassicae on different foliage components of canola and mustard rape.

Revisiting Sustainability of Fungicide Seed Treatments for Field Crops.

The use of fungicide seed treatment (FST) is a very common practice worldwide. The purported effectiveness of many fungicides in providing broad-spectrum and systemic control of important diseases and the perception that FST reduces overall pesticide use, hence lowering environmental impacts, have greatly promoted the use of FST in the last five decades. Since there have been rapid advancements in the types, formulations, and application methods for seed treatments, there is a need to re-evaluate the benefits versus the risks of FST as a practice. While the use of seeds treated with neonicotinoid insecticides has come under scrutiny due to concern over potential nontarget effects, there are knowledge gaps on potential negative impacts of FST on operators' (those who apply, handle, and use treated seeds) health and nontarget soil organisms (both macro- and microorganisms). Here we review existing knowledge on key fungicides used for seed treatments, benefits and risks related to FST, and propose recommendations to increase benefits and limit risks related to the use of FST. We found FST is applied to almost 100% of sown seeds for the most important arable crops worldwide. Fungicides belonging to 10 chemical families and with one or several types of mobility (contact, locally systemic, and xylem mobile) are used for seed treatment, although the majority are xylem mobile. Seed treatments are applied by the seed distributor, the seed company, and the farmer, although the proportion of seed lots treated by these three groups vary from one crop to another. The average quantity of fungicide active ingredient (a.i.) applied via seed treatment depends on the crop species, environment(s) into which seed is planted, and regional or local regulations. Cost-effectiveness, protection of the seed and seedlings from pathogens up to 4-5 weeks from sowing, user friendliness, and lower impact on human health and nontarget soil organisms compared with foliar spray and broadcast application techniques, are among the most claimed benefits attributed to FST. In contrast, inconsistent economic benefits, development of resistance by soilborne pathogens to many fungicides, exposure risks to operators, and negative impacts on nontarget soil organisms are the key identified risks related to FST. We propose eight recommendations to reduce risks related to FST and to increase their benefits.

Understanding Why Effective Fungicides Against Individual Soilborne Pathogens Are Ineffective with Soilborne Pathogen Complexes.

Annual forage legumes across southern Australia continue to be devastated by soilborne diseases. Nine fungicide seed treatments (thiram, metalaxyl, iprodione, phosphonic acid, propamocarb, fluquinconazole, difenoconazole + metalaxyl, ipconazole + metalaxyl, sedaxane + difenoconazole + metalaxyl) and four foliar fungicide treatments (phosphonic acid, metalaxyl, propamocarb, iprodione) were tested on four subterranean clover cultivars against individual oomycete soilborne pathogens Pythium irregulare, Aphanomyces trifolii, and Phytophthora clandestina and the fungal pathogen Rhizoctonia solani. Best treatments were then further tested across southern Australia in 2 years of field experiments. Under controlled conditions, seed treatment with thiram was best against damping-off caused by P. irregulare across the four cultivars (Woogenellup, Riverina, Seaton Park, Meteora), while metalaxyl was the most effective for maximizing root and shoot weights. Against A. trifolii, metalaxyl, iprodione, difenoconazole + metalaxyl, ipconazole + metalaxyl, and sedaxane + difenoconazole + metalaxyl, all reduced damping-off; sedaxane + difenoconazole + metalaxyl, fluquinconazole, and ipconazole + metalaxyl all reduced lateral root disease across two or more cultivars; while iprodione, thiram, and sedaxane + difenoconazole + metalaxyl increased shoot dry weight. Against P. clandestina, metalaxyl was the most effective in reducing tap and lateral root rot followed by ipconazole + metalaxyl or phosphonic acid for tap and lateral rot, respectively. Against R. solani, there were no effects of fungicides. For P. irregulare and P. clandestina, there were strong seed fungicide × cultivar interactions (P < 0.001). Under controlled conditions for foliar fungicide spray treatments, phosphonic acid was best at preventing productivity losses from A. trifolii, but was ineffective against P. clandestina, P. irregulare, or R. solani. Overall, controlled environment studies highlighted strong potential for utilizing seed treatments against individual pathogens to ensure seedling emergence and early survival, with seed and foliar sprays enhancing productivity by reducing seedling damping-off and root disease from individual pathogens. However, in field experiments over 2 years across southern Australia against naturally occurring soilborne pathogen complexes involving these same pathogens, only rarely did fungicide seed treatments or foliar sprays tested show any benefit. It is evident that currently available fungicide seed and/or foliar spray treatment options do not offer effective field mitigation of damping-off and root disease on annual forage legumes that underpin livestock production across southern Australia. The main reason for this failure relates to the unpredictable and ever-changing soilborne pathogen complexes involved, highlighting a need to now refocus away from fungicide options, particularly toward developing and deploying new host tolerances, but also in deploying appropriate cultural control options.

Comparative Reaction of Camelina sativa to Sclerotinia sclerotiorum and Leptosphaeria maculans.

Sclerotinia sclerotiorum and Leptosphaeria maculans are two of the most important pathogens of many cruciferous crops. The reaction of 30 genotypes of Camelina sativa (false flax) was determined against both pathogens. C. sativa genotypes were inoculated at seedling and adult stages with two pathotypes of S. sclerotiorum, highly virulent MBRS-1 and less virulent WW-1. There were significant differences (P < 0.001) among genotypes, between pathotypes, and a significant interaction between genotypes and pathotypes in relation to percent cotyledon disease index (% CDI) and stem lesion length. Genotypes 370 (% CDI 20.5, stem lesion length 1.8 cm) and 253 (% CDI 24.8, stem lesion length 1.4 cm) not only consistently exhibited cotyledon and stem resistance, in contrast to susceptible genotype 2305 (% CDI 37.7, stem lesion length 7.2 cm), but their resistance was independent to S. sclerotiorum pathotype. A F5-recombinant inbred line population was developed from genotypes 370 × 2305 and responses characterized. Low broad-sense heritability indicated a complex pattern of inheritance of resistance to S. sclerotiorum. Six isolates of L. maculans, covering combinations of five different avirulent loci (i.e., five different races), were tested on C. sativa cotyledons across two experiments. There was a high level of resistance, with % CDI < 17, and including development of a hypersensitive reaction. This is the first report of variable reaction of C. sativa to different races of L. maculans and the first demonstrating comparative reactions of C. sativa to S. sclerotiorum and L. maculans. This study not only provides new understanding of these comparative resistances in C. sativa, but highlights their potential as new sources of resistance, both for crucifer disease-resistance breeding in general and to enable broader adoption of C. sativa as a more sustainable oilseed crop in its own right.

Inert Materials as Long-Term Carriers and Disseminators of Viable Leptosphaeria maculans Ascospores and Wider Implications for Ascomycete Pathogens.

The viability of ascospores of the Phoma stem canker (blackleg) pathogen, Leptosphaeria maculans, was tested on a range of carrier materials, including metals, fabrics, woods, and plastics, and under different temperature conditions of 23 and 4, 36 and 14, and 45 and 15°C day and night, respectively. At 23 and 4°C (day and night, respectively), ascospores remained viable for up to 240 days on Tasmanian oak (Eucalyptus regnans) and pine wood (Pinus radiata). At 36 and 14°C (day and night, respectively), ascospores remained viable on pine wood for up to 180 days. At 45 and 15°C (day and night, respectively), ascospores remained viable up to 60 days on jute. There were also significant differences (P < 0.001) between carrier materials in their abilities to retain ascospores following washing. At least 30% of intact ascospores recovered from inert carrier materials were able to germinate on artificial growth media within 48 h of recovery and some ascospores were still viable after 240 days. These findings confirm that L. maculans ascospores remain viable for a much longer time in the absence of a host than previously considered. This demonstrates the importance of inert materials as long-term and long-distance carriers of viable L. maculans ascospores, and highlights their potential role for spread of L. maculans races to new regions and countries via farming equipment, clothing, and other associated materials. Local, national, and international biosecurity agencies need to be aware that the risks of spread of ascomycete plant, animal, and human pathogens via inert materials are significantly greater than currently assessed.

Virome Characterization of a Collection of S. sclerotiorum from Australia.

Sclerotinia sclerotiorum is a devastating plant pathogen that attacks numerous economically important broad acre and vegetable crops worldwide. Mycoviruses are widespread viruses that infect fungi, including S. sclerotiorum. As there were no previous reports of the presence of mycoviruses in this pathogen in Australia, studies were undertaken using RNA_Seq analysis to determine the diversity of mycoviruses in 84 Australian S. sclerotiorum isolates collected from various hosts. After RNA sequences were subjected to BLASTp analysis using NCBI database, 285 contigs representing partial or complete genomes of 57 mycoviruses were obtained, and 34 of these (59.6%) were novel viruses. These 57 viruses were grouped into 10 distinct lineages, namely Endornaviridae (four novel mycoviruses), Genomoviridae (isolate of SsHADV-1), Hypoviridae (two novel mycoviruses), Mononegavirales (four novel mycovirusess), Narnaviridae (10 novel mycoviruses), Partitiviridae (two novel mycoviruses), Ourmiavirus (two novel mycovirus), Tombusviridae (two novel mycoviruses), Totiviridae (one novel mycovirus), Tymovirales (five novel mycoviruses), and two non-classified mycoviruses lineages (one Botrytis porri RNA virus 1, one distantly related to Aspergillus fumigatus tetramycovirus-1). Twenty-five mitoviruses were determined and mitoviruses were dominant in the isolates tested. This is not only the first study to show existence of mycoviruses in S. sclerotiorum in Australia, but highlights how they are widespread and that many novel mycoviruses occur there. Further characterization of these mycoviruses is warranted, both in terms of exploring these novel mycoviruses for innovative biocontrol of Sclerotinia diseases and in enhancing our overall knowledge on viral diversity, taxonomy, ecology, and evolution.