Inter- and Intra-Pathogen Interactions Emanating from Coinfection with Different Fungal and Viral strains in Canola Cultivars with Differing Host Resistances.

Few recent investigations examine coinfection interactions between fungal and viral plant pathogens. Here, we investigated coinfections between Leptosphaeria maculans and turnip mosaic virus (TuMV) in canola (Brassica napus). Different combinations of L. maculans isolate P11 and resistance breaking isolates L. maculans UWA192 and TuMV 12.1, were inoculated to three cultivars with differing pathogen resistances/susceptibilities. They were inoculated first to entire or half cotyledons 10-12 days after emergence, and second to opposite entire or half cotyledons on the same day (day 0) or 3 or 7 days afterwards. The parameters measured were L. maculans cotyledon disease index (%CDI), and TuMV systemically infected leaf symptom intensity (SI) and virus concentration (VC). Except when both day 0 inoculations were with isolate UWA192, %CDI values were supressed strongly or only weakly when isolates P11 and/or UWA192 were inoculated to plants with L. maculans single gene resistance (SGR) or polygenic resistance, respectively. However, except when isolate P11 was inoculated first and UWA192 second, these values declined after inoculation day 0 when SGR was absent. TuMV infection suppressed %CDI values, although this decrease was usually smaller following day 0 half cotyledon inoculations. When TuMV temperature sensitive extreme resistance was present and both inoculations were with TuMV, SI and VC values diminished greatly. However, the extent of this decrease was reduced when second inoculations were with L. maculans. SI and VC values were also smaller when SGR was present and second inoculations were with L. maculans. When L. maculans resistance was lacking, SI and VC values were smaller when second inoculations to entire cotyledons were with L. maculans rather than TuMV. This also occurred after second half cotyledon inoculations with isolate P11 but not isolate UWA192. Therefore, diverse inter- or intra-pathogen interactions developed depending upon host resistance, isolate combination, cotyledon inoculation approach and second inoculation timing.

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.). A total of 236 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 (totaling ∼4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, 2 accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totaling ∼10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, 1 L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, Walkaway-3 (WW-3; pathotype 10). Against this isolate, five L. angustifolius accessions showed an HR response and four showed an R response, and the L. digitatus accession showed a moderate resistance 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. cosentinii 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 an HR response, and 146 (∼45%) showed an 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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Attack modes and defence reactions in pathosystems involving Sclerotinia sclerotiorum, Brassica carinata, B. juncea and B. napus.

BACKGROUND AND AIMS: Sclerotinia stem rot (SSR, Sclerotinia sclerotiorum) is a damaging disease of oilseed brassicas world-wide. Host resistance is urgently needed to achieve control, yet the factors that contribute to stem resistance are not well understood. This study investigated the mechanisms of resistance to SSR. METHODS: Stems of 5-week-old Brassica carinata, B. juncea and B. napus of known resistance were infected via filter paper discs impregnated with S. sclerotiorum mycelium under controlled conditions. Transverse sections of the stem and portions of the stem surface were examined using optical and scanning electron microscopy. The association of anatomical features with the severity of disease (measured by mean lesion length) was determined. KEY RESULTS: Several distinct resistance mechanisms were recorded for the first time in these Brassica-pathogen interactions, including hypersensitive reactions and lignification within the stem cortex, endodermis and in tissues surrounding the lesions. Genotypes showing a strong lignification response 72 h post-infection (hpi) tended to have smaller lesions. Extensive vascular invasion by S. sclerotiorum was observed only in susceptible genotypes, especially in the vascular fibres and xylem. Mean lesion length was negatively correlated with the number of cell layers in the cortex, suggesting progress of S. sclerotiorum is impeded by more cell layers. Hyphae in the centre of lesions became highly vacuolate 72 hpi, reflecting an ageing process in S. sclerotiorum hyphal networks that was independent of host resistance. The infection process of S. sclerotiorum was analogous in B. carinata and B. napus. Infection cushions of the highly virulent isolate of S. sclerotiorum MBRS-1 were grouped together in dense parallel bundles, while hyphae in the infection cushions of a less aggressive isolate WW-3 were more diffuse, and this was unaffected by host genotype. CONCLUSIONS: A variety of mechanisms contribute to host resistance against S. sclerotiorum across the three Brassica species. These complex interactions between pathogen and host help to explain variable expressions of resistance often observed in the field.

Cercosporin From Pseudocercosporella capsellae and its Critical Role in White Leaf Spot Development.

Pseudocercosporella capsellae, the causative agent of white leaf spot disease in Brassicaceae, can produce a purple-pink pigment on artificial media resembling, but not previously confirmed as, the toxin cercosporin. Chemical extraction with ethyl acetate from growing hyphae followed by quantitative (thin-layer chromatography [TLC] and high-performance liquid chromatography [HPLC]) and qualitative methods showed an identical absorption spectrum, with similar retardation factor (Rf) values on TLC papers and an identical peak with the same retention time in HPLC as for a standard for cercosporin. We believe this is the first report to confirm that the purple-pink pigment produced by P. capsellae is cercosporin. Confocal microscopy detected green autofluorescence of cercosporin-producing hyphae, confirming the presence of cercosporin inside hyphae. The highly virulent UWA Wlra-7 isolate of P. capsellae produced the greatest quantity of cercosporin (10.69 mg g-1). The phytotoxicity and role of cercosporin in disease initiation across each of three Brassicaceae host species (Brassica juncea, B. napus, and Raphanus raphanistrum) was also studied. Culture filtrates containing cercosporin were phytotoxic to all three host plant species, producing large, white lesions on highly sensitive B. juncea, only water-soaked areas on least sensitive R. raphanistrum, and intermediate lesions on B. napus. It is noteworthy that sensitivity to cercosporin of these three host species was analogous to their susceptibility to the pathogen, viz., B. juncea the most susceptible, R. raphanistrum the least susceptible, and B. napus intermediate. The presence of cercosporin in the inoculum significantly increased disease severity on the highly cercosporin-sensitive B. juncea. We believe that this is the first study to demonstrate that P. capsellae produces cercosporin in liquid culture rather than agar media. Finally, this study highlights an important role of cercosporin as a pathogenicity factor in white leaf spot disease on Brassicaceae as evidenced by the ability of the cercosporin-rich culture filtrate to reproduce white leaf spot lesions on host plants and by the enhanced virulence of P. capsellae in the presence of cercosporin.

Relative Host Resistance to Black Spot Disease in Field Pea (Pisum sativum) is Determined by Individual Pathogens.

Black spot, also known as Ascochyta blight, is the most important disease on field pea (Pisum sativum). It is caused by a complex of pathogens, the most important of which in Australia include Didymella pinodes, Phoma pinodella, and P. koolunga. The relative proportions of these and other component pathogens of the complex fluctuate widely across time and geographic locations in Australia, limiting the ability of breeders to develop varieties with effective resistance to black spot. To address this, 40 field pea genotypes were tested under controlled environment conditions for their individual stem and leaf responses against these three pathogens. Disease severity was calculated as area under disease progress curve (AUDPC), and subsequently converted to mean rank (MR). The overall rank (OR) for each pathogen was used to compare response of genotypes under inoculation with each pathogen. The expressions of host resistance across the field pea genotypes were largely dependent upon the individual test pathogen and whether the test was on stem or leaf. Overall, P. koolunga caused most severe stem disease; significantly more severe than either D. pinodes or P. pinodella. This is the first report of the host resistance identified in field pea to P. koolunga; the five genotypes showing highest resistance on stem, viz. 05P778-BSR-701, ATC 5338, ATC 5345, Dundale, and ATC 866, had AUDPC MR values <250.4, while the AUDPC MR values of the 19 genotypes showing the best resistance on leaf was less than 296.8. Two genotypes, ATC 866 and Dundale, showed resistance against P. koolunga on both stem and leaf. Against D. pinodes, the four and 16 most resistant genotypes on stem and leaf had AUDPC MR values <111.2 and <136.6, respectively, with four genotypes showing resistance on both stem and leaf including 05P770-BSR-705, Austrian Winter Pea, 06P822-(F5)-BSR-6, and 98107-62E. Against P. pinodella, four and eight genotypes showing the best resistance on stem and leaf had AUDPC MR values <81.3 and <221.9, respectively; three genotypes, viz. 98107-62E, Dundale, and Austrian Winter Pea showed combined resistance on stem and leaf. A few genotypes identified with resistance against two major pathogens of the complex will be of particular significance to breeding programs. These findings explain why field pea varieties arising from breeding programs in Australia fail to display the level or consistency of resistance required against black spot and why there needs to be a wider focus than D. pinodes in breeding programs.

Effect of Timing and Duration of Soil Saturation on Soilborne Pythium Diseases of Common Bean (Phaseolus vulgaris).

Understanding combined abiotic (waterlogging) and biotic (Pythium spp.) stress resistance remains an important challenge to improving common bean (Phaseolus vulgaris) productivity in disease-prone regions with irregular but intensive rainfall patterns. This study documented the effects of timing (1, 3, 5, 7, and 9 days after sowing) and duration (3, 6, 12, and 24 h) of soil saturation (waterlogging) on damping-off, as well as hypocotyl and root diseases of common bean caused by Pythium irregulare. There were significant effects of timing of waterlogging as well as the presence or absence of the pathogen on emergence of the three bean varieties tested; namely, 'Gourmet Delight', 'Brown Beauty', and 'Pioneer'. The interaction between time of waterlogging and variety was significant for both root and hypocotyl disease severities. In the presence of P. irregulare, waterlogging 1 day after sowing resulted in the least emergence (55.2 ± 5.6%), although plants that survived after 5 weeks had less hypocotyl and root disease (percent hypocotyl disease index [%HDI] ± standard deviation [SD] = 42.0 ± 2.1% and percent root disease index [%RDI] ± SD = 42.4 ± 2.1%, respectively) than nonwaterlogged plants (%HDI = 50.8 ± 2.1% and %RDI = 48.0 ± 2.1%, respectively). The most severe disease assessed 5 weeks after sowing occurred when plants were subjected to waterlogging 9 days after sowing (%HDI = 61.3 ± 2.1% and %RDI = 56.0 ± 2.1%). In general, both hypocotyl and root disease severity increased as the duration of waterlogging increased from 1 to 24 h, with %HDI increasing from 53.9 ± 3.2% to 70.9 ± 3.2%, while %RDI increased from 57.2 ± 1.5% to 73.7 ± 1.5%. Varieties responded differentially in terms of disease development after waterlogging, with the least hypocotyl and root disease on Gourmet Delight (%HDI = 51.4 ± 3.2 and %RDI = 60.1 ± 1.5, respectively) and greatest on Pioneer (%HDI = 66.2 ± 3.2 and %RDI = 64.9 ± 1.5, respectively). Despite being susceptible to hypocotyl and root disease, Pioneer had the greatest emergence and shoot dry weight overall among the three varieties, suggesting that this variety has a degree of tolerance to waterlogging, P. irregulare infection, and the combination of these two stresses. Although the resistance of Gourmet Delight could be exploited to breed bean varieties that exhibit less hypocotyl and root disease when waterlogging occurs, the tolerance to both P. irregulare infection and waterlogging observed for Pioneer could also be exploited to breed varieties that incur less damage from hypocotyl or root disease or waterlogging. Furthermore, this study demonstrated what appears to be independent resistance to hypocotyl versus root infection by P. irregulare, which offers an opportunity to combine resistance to both stresses to reduce the impact of damping-off and root rot in conditions conducive for P. irregulare.

Species of Pythium Associated with Seedling Root and Hypocotyl Disease on Common Bean (Phaseolus vulgaris) in Western Australia.

The occurrence and distribution of Pythium spp. were determined by collecting isolates of Pythium from common bean (Phaseolus vulgaris) plants showing root or hypocotyl disease symptoms from different areas of Western Australia in 2012. Eight different Pythium species (Pythium conidiophorum, P. diclinum, P. intermedium, P. irregulare, P. lutarium, P. mamillatum, P. pachycaule, and P. perplexum) were isolated and identified according to molecular sequences. P. irregulare was the most widespread Pythium sp. All species, except P. perplexum, were pathogenic to the hypocotyl and root of common bean. We believe this is the first report of P. intermedium as a pathogen on common bean worldwide. This is also the first report of P. conidiophorum, P. intermedium, P. lutarium, P. mamillatum, P. pachycaule, and P. diclinum as pathogens on common bean in Australia and the first report of P. irregulare as a pathogen on common bean in Western Australia. P. intermedium was the most pathogenic species, causing the most severe disease on 'Gourmet Delight' (percent root disease index [%RDI] 75 ± 2.9 and percent hypocotyl disease index [%HDI] 59.2 ± 3.2) and 'Pioneer' (%RDI 75 ± 2.9 and %HDI 65.8 ± 3.2). That the relative susceptibility or resistance (the ability of a plant to reduce the extent of invasion by the pathogen) of a given bean variety to one Pythium sp. was, in general, similar across the other Pythium spp. was an important finding, because this opens up opportunities to utilize a single virulent isolate of one Pythium sp. to identify general resistance to a wider spectrum of Pythium spp.