The soil bacterial community regulates germination of Plasmodiophora brassicae resting spores rather than root exudates.

Clubroot, caused by Plasmodiophora brassicae, is a severe soil-borne disease that restricts the production of cruciferous crops worldwide. A better understanding of biotic and abiotic factors regulating germination of P. brassicae resting spores in the soil is significant for developing novel control methods. Previous studies reported that root exudates can trigger P. brassicae resting spore germination, thus enabling a targeted attack of P. brassicae on host plant roots. However, we found that native root exudates collected under sterile conditions from host or non-host plants cannot stimulate the germination of sterile spores, indicating that root exudates may not be direct stimulation factors. Instead, our studies demonstrate that soil bacteria are essential for triggering germination. Through 16s rRNA amplicon sequencing analysis, we found that certain carbon sources and nitrate can reshape the initial microbial community to an inducing community leading to the germination of P. brassicae resting spores. The stimulating communities significantly differed in composition and abundance of bacterial taxa compared to the non-stimulating ones. Several enriched bacterial taxa in stimulating community were significantly correlated with spore germination rates and may be involved as stimulation factors. Based on our findings, a multi-factorial 'pathobiome' model comprising abiotic and biotic factors is proposed to represent the putative plant-microbiome-pathogen interactions associated with breaking spore dormancy of P. brassicae in soil. This study presents novel views on P. brassicae pathogenicity and lays the foundation for novel sustainable control strategies of clubroot.

The interplay of suppressive soil bacteria and plant root exudates determines germination of microsclerotia of Verticillium longisporum.

Dormant microsclerotia play a vital role in the survival and spread of Verticillium longisporum, as they can stay viable in the soil and maintain their infectivity for many years. In our previous work, we revealed that soil bacterial volatiles are a key inhibitory factor causing microsclerotia dormancy in the soil. In this study, we further demonstrate that root exudates collected from both host and non-host plants can effectively rescue microsclerotia from bacterial suppression and initiate germination. To identify the specific compounds in root exudates responsible for microsclerotia germination, we fractionated the collected root exudates into polar and non-polar compounds. Subsequently, we conducted comprehensive bioassays with each fraction on germination-suppressed microsclerotia. The result revealed a pivotal role of primary metabolites in root exudates, particularly glutamic acid, in triggering microsclerotia germination and overcoming bacterial inhibition. Moreover, our studies revealed a decrease in inhibitory bacterial volatile fatty acids when bacteria were cultured in the presence of root exudates or glutamic acid. This suggests a potential mechanism, by which root exudates set-off bacterial suppression on microsclerotia. Here, we reveal for the first time that plant root exudates, instead of directly inducing the germination of microsclerotia, enact a set-off effect by counteracting the suppressive impact of soil bacteria on the microsclerotia germination process. This nuanced interaction advances our understanding of the multifaceted dynamics governing microsclerotia dormancy and germination in the soil environment. IMPORTANCE: Our research provides first-time insights into the crucial interaction between plant root exudates and soil bacteria in regulating the germination of Verticillium longisporum microsclerotia, a significant structure in the survival and proliferation of this soil-borne pathogen. We describe so far unknown mechanisms, which are key to understand how root infections on oilseed rape can occur. By pinpointing primary metabolites in root exudates as key factors in overcoming bacteria-induced dormancy and promote microsclerotia germination, our study highlights the potential for exploiting plant - as well as soil microbe-derived - compounds to control V. longisporum. This work underscores the importance of elucidating the nuanced interactions within the soil ecosystem to devise innovative strategies for managing root infective plant diseases, thereby contributing to the resilience and health of cropping systems.

Photosynthetic Costs and Impact on Epidemiological Parameters Associated with Ht Resistance Genes in Maize Lines Infected with Exserohilum turcicum.

Northern corn leaf blight, caused by Exserohilum turcicum, is mainly controlled by the use of resistant cultivars. Maize lines carrying individual resistance genes B37Ht1, B37Ht2, B37Ht3, and B37Htn1 express different defense symptoms having an impact on the photosynthetic activity, the accumulation of reactive oxygen species, and epidemiological parameters. Plants were inoculated with a race 0 isolate of E. turcicum conferring a compatible interaction with B37 and incompatible interactions with plants carrying resistance genes. Five days postinoculation (dpi), the resistant lines displayed a reduction in leaf CO2 assimilation of 30 to 80% compared with healthy plants. At 14 dpi, inoculated plants of B37Ht1 showed a significant decrease in leaf CO2 assimilation, similar to B37 (up to 94%). The instantaneous carboxylation efficiency was significantly reduced on inoculated plants of the lines B37Ht2, B37Ht3, and B37Htn1 (54 to 81%) at 5 dpi. Curiously, the reduction in carboxylation efficiency for B37 and B37Ht1 (up to 95%) was higher at 14 dpi than at 5 dpi (up to 81%). At 6 dpi, low levels of H2O2 were detected in B37Ht1, in contrast to B37Htn1, where a high H2O2 level and peroxidase activity were observed. The sporulation rate on B37Ht1, B37Ht3, and B37Htn1 decreased by 92% compared with the susceptible control, whereas strong sporulation occurred in lesions on line B37Ht2. The resistance in maize to E. turcicum conferred by Ht resistance genes is associated with photosynthetic costs and may have quite contrasting effects on host physiology and major epidemiological parameters, such as sporulation, which contributes inoculum for secondary infections.

Lignin Composition and Timing of Cell Wall Lignification Are Involved in Brassica napus Resistance to Stem Rot Caused by Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) is an economically and globally significant disease in oilseed rape (Brassica napus) caused by the necrotrophic ascomycete Sclerotinia sclerotiorum. This study explored the role of cell wall reinforcement by lignin as a relevant factor for effective plant defense against attack by this pathogen. Expression of key genes in the phenylpropanoid pathway and the induced synthesis of lignin in infected stem tissues were investigated in a study comparing a susceptible ('Loras') and a moderately resistant cultivar ('Zhongyou 821' [ZY821]). Data revealed an earlier and more rapid defense activation in ZY821 through upregulation of transcript levels of genes related to key steps in the phenylpropanoid pathway associated with increased lignin deposition in the resistant B. napus genotype. Expression level of BnCAD5, encoding a cinnamyl alcohol dehydrogenase, responsible for conversion of monolignol to lignin, was more rapidly upregulated in ZY821 than 'Loras'. The similar expression pattern of BnCAD5 and the gene BnF5H, encoding for ferulate-5-hydroxylase, which catalyzes the synthesis of syringyl (S) lignin precursors, suggests that BnCAD5 is involved in S lignin formation. Histological observations confirmed these results, showing an earlier increase of S lignin deposition in the infected resistant genotype. Deposition of guaiacyl lignin was detected in both genotypes and is thus considered a component of basal, cultivar-independent defense response of B. napus to stem rot. The results indicate the importance of cell wall modification for quantitative stem rot resistance by responses in the phenylpropanoid metabolism generating distinct lignin types on different temporal scales.

Dynamic Insertion of Pot3 in AvrPib Prevailing in a Field Rice Blast Population in the Philippines Led to the High Virulence Frequency Against the Resistance Gene Pib in Rice.

The Magnaporthe oryzae avirulence gene AvrPib is required for the resistance mediated by its cognate resistance gene Pib, which has been intensively used in indica rice breeding programs in many Asian countries. However, the sequence diversity of AvrPib among geographically distinct M. oryzae populations was recently shown to be increasing. Here, we selected a field population consisting of 248 rice blast isolates collected from a disease hotspot in Philippine for the analysis of AvrPib haplotypes and their pathogenicity against Pib. We found that all of the isolates were virulent to Pib and each of them contained an insertion of Pot3 transposon in AvrPib. Moreover, Pot3 insertion was detected in different genomic positions, resulting in three different AvrPib haplotypes, designated avrPib-H1 to H3. We further conducted a genome-wide Pot2 fingerprinting analysis by repetitive element palindromic polymerase chain reaction (PCR) and identified seven different lineages out of 47 representative isolates. The isolates belonging to the same lineage often had the same AvrPib haplotype. In contrast, the isolates having the same AvrPib haplotypes did not always belong to the same lineages. Both mating types MAT1-1 and MAT1-2 were identified in the population in Bohol and the latter appeared dominant. On the host side, we found that 32 of 52 released rice varieties in the Philippines contained Pib diagnosed by PCR gene-specific primers and DNA sequencing of gene amplicons, suggesting that it was widely incorporated in different rice varieties. Our study highlights the genetic dynamics of rice blast population at both the AvrPib locus and the genome-wide levels, providing insight into the mechanisms of the mutations in AvrPib leading to the breakdown of Pib-mediated resistance in rice.

Potential for Seed Transmission of Verticillium longisporum in Oilseed Rape (Brassica napus).

Verticillium longisporum is a soilborne vascular fungal pathogen that has spread throughout the European oilseed rape cultivation area since the 1980s and was detected in canola fields in Canada in 2014. In a series of greenhouse and field inoculation experiments using V. longisporum-resistant and susceptible cultivars of winter and spring types of oilseed rape, the present study investigated the potential of V. longisporum dissemination by seeds of Brassica napus. Greenhouse inoculation studies with a DsRed-labeled isolate of V. longisporum confirmed the systemic growth of the pathogen from roots to seeds. Further monitoring of plant colonization in the greenhouse with a species-specific real-time polymerase chain reaction assay verified the pathogen growth from roots to stem bases, pods, and seeds in root-inoculated plants. The frequency of recovery of viable colonies of V. longisporum from seeds harvested from greenhouse-grown inoculated plants ranged from 0.08 to 13.3%. The frequency of seed transmission in the greenhouse differed in oilseed rape cultivars varying in susceptibility to V. longisporum. Subsequent studies on transmission of the disease into the offspring revealed that only 1.7 to 2.3% of plants showed disease symptoms as confirmed by the formation of microsclerotia in the stems. Results from field-grown plants differed from the greenhouse studies. The degree of seed transmission in the field was dependent on the crop type. Although only low concentrations of DNA of V. longisporum were detectable in seeds harvested from severely infected winter oilseed rape, significantly greater concentrations of fungal DNA were found in seeds of spring-type oilseed rape, at similar soil conditions and inoculum densities. Correspondingly, plating seeds that were harvested from infected plants on agar yielded viable V. longisporum colonies only from seeds of the spring-type but not of the winter-type plants. Lack of seed infection in the winter-type crop was confirmed in two seasons. Equally, none of the offspring grown from seeds from severely diseased winter oilseed rape plants developed symptoms of Verticillium stem striping. The results suggest that the rate of seed transmission of V. longisporum depends on the degree of plant colonization, which is significantly faster under greenhouse than field conditions and in a spring-sown crop compared with an autumn-sown oilseed rape crop. According to our studies, disease transmission by seeds from European winter oilseed rape production cannot be confirmed.

Contrasting Patterns of Colonization with Verticillium longisporum in Winter- and Spring-Type Oilseed Rape (Brassica napus) in the Field and Greenhouse and the Role of Soil Temperature.

Oilseed rape, an important source of vegetable plant oil, is threatened by Verticillium longisporum, a soil-borne vascular fungal pathogen so far occurring in oilseed rape growing regions in Europe and Canada. Despite intensive research into V. longisporum in the last decades in controlled conditions, basic knowledge is still lacking about the time course of infection, temporal pattern of colonization, and disease development on field-grown plants. In this study, colonization of roots, stem bases, and stems with V. longisporum was followed by real-time PCR from the seedling until mature plant stages in 2-year field experiments with microsclerotia-infested plots and either spring-type or autumn-sown (winter-type) oilseed rape cultivars. The temporal pattern of plant colonization differed between greenhouse and field-grown oilseed rape and between spring- and winter-type plants in the field. Within 28 to 35 days, a continuous systemic colonization with V. longisporum was detected in roots and shoots of young plants in the greenhouse associated with significant stunting. In contrast, real-time PCR analysis of V. longisporum in field-grown winter oilseed rape plants displayed a strongly discontinuous colonization pattern with low fungal growth in roots during juvenile growth stages until flowering, whereas in spring oilseed rape, no root colonization was observed until early flowering stages. Hence, stem colonization with the pathogen required 6 months in winter oilseed rape and 1 month in spring oilseed rape from the time of initial root infection. The different patterns of stem colonization were related to soil temperature. Average soil temperatures in 5-cm depth during 7 days before sampling time points from 2 years of field experiments displayed a significant relationship with fungal colonization in the root. A climate chamber inoculation trial with soil temperature levels that varied from 6 to 18°C revealed a threshold temperature of >12°C in the soil to enable root invasion. This soil condition is reached in winter-type oilseed rape in the field in Germany either until the eight-leaf stage in early autumn or after pod stage in spring, whereas in spring-sown oilseed rape early root infection is delayed owing to the cool conditions during juvenile growth stages. The delay of stem colonization in field-grown oilseed rape may explain the lack of stunting as observed in the greenhouse and the previously reported inconsistent effects of V. longisporum on yield levels and seed quality, which were confirmed in this study.

Finding invisible quantitative trait loci with missing data.

Evolutionary processes during plant polyploidization and speciation have led to extensive presence-absence variation (PAV) in crop genomes, and there is increasing evidence that PAV associates with important traits. Today, high-resolution genetic analysis in major crops frequently implements simple, cost-effective, high-throughput genotyping from single nucleotide polymorphism (SNP) hybridization arrays; however, these are normally not designed to distinguish PAV from failed SNP calls caused by hybridization artefacts. Here, we describe a strategy to recover valuable information from single nucleotide absence polymorphisms (SNaPs) by population-based quality filtering of SNP hybridization data to distinguish patterns associated with genuine deletions from those caused by technical failures. We reveal that including SNaPs in genetic analyses elucidate segregation of small to large-scale structural variants in nested association mapping populations of oilseed rape (Brassica napus), a recent polyploid crop with widespread structural variation. Including SNaP markers in genomewide association studies identified numerous quantitative trait loci, invisible using SNP markers alone, for resistance to two major fungal diseases of oilseed rape, Sclerotinia stem rot and blackleg disease. Our results indicate that PAV has a strong influence on quantitative disease resistance in B. napus and that SNaP analysis using cost-effective SNP array data can provide extensive added value from 'missing data'. This strategy might also be applicable for improving the precision of genetic mapping in many important crop species.

Growth of Verticillium longisporum in Xylem Sap of Brassica napus is Independent from Cultivar Resistance but Promoted by Plant Aging.

As Verticillium stem striping of oilseed rape (OSR), a vascular disease caused by Verticillium longisporum, is extending into new geographic regions and no control with fungicides exists, the demand for understanding mechanisms of quantitative resistance increases. Because V. longisporum is strictly limited to the xylem and resistance is expressed in the systemic stage post root invasion, we investigated a potential antifungal role of soluble constituents and nutritional conditions in xylem sap as determinants of cultivar resistance of OSR to V. longisporum. Assessment of biometric and molecular genetic parameters applied to describe V. longisporum resistance (net area under disease progress curve, stunting, stem thickness, plant biomass, and V. longisporum DNA content) showed consistent susceptibility of cultivar 'Falcon' in contrast to two resistant genotypes, 'SEM' and 'Aviso'. Spectrophotometric analysis revealed a consistently stronger in vitro growth of V. longisporum in xylem sap extracted from OSR compared with the water control. Further comparisons of fungal growth in xylem sap of different cultivars revealed the absence of constitutive or V. longisporum induced antifungal activity in the xylem sap of resistant versus susceptible genotypes. The similar growth of V. longisporum in xylem sap, irrespective of cultivar, infection with V. longisporum and xylem sap filtration, was correlated with about equal amounts of total soluble proteins in xylem sap from these treatments. Interestingly, compared with younger plants, xylem sap from older plants induced significantly stronger fungal growth. Growth enhancement of V. longisporum in xylem sap of aging plants was reflected by increased contents of carbohydrates, which was consistent in mock or V. longisporum-infected plants and independent from cultivar resistance. The improved nutritional conditions in the xylem of more mature plants may explain the late appearance of disease symptoms, which are observed only in late maturity stages of plants in the field. While falsifying the presence of antifungal activity in xylem sap of resistant cultivars, this study strengthens previous findings that indicated a significant role of physical cell wall bound resistance factors involved in quantitative, cultivar-related resistance of B. napus to V. longisporum.

The Vascular Pathogen Verticillium longisporum Does Not Affect Water Relations and Plant Responses to Drought Stress of Its Host, Brassica napus.

Verticillium longisporum is a host-specific vascular pathogen of oilseed rape (Brassica napus L.) that causes economic crop losses by impairing plant growth and inducing premature senescence. This study investigates whether plant damage through Verticillium stem striping is due to impaired plant water relations, whether V. longisporum affects responses of a susceptible B. napus variety to drought stress, and whether drought stress, in turn, affects plant responses to V. longisporum. Two-factorial experiments on a susceptible cultivar of B. napus infected or noninfected with V. longisporum and exposed to three watering levels (30, 60, and 100% field capacity) revealed that drought stress and V. longisporum impaired plant growth by entirely different mechanisms. Although both stresses similarly affected plant growth parameters (plant height, hypocotyl diameter, and shoot and root dry matter), infection of B. napus with V. longisporum did not affect any drought-related physiological or molecular genetic plant parameters, including transpiration rate, stomatal conductance, photosynthesis rate, water use efficiency, relative leaf water content, leaf proline content, or the expression of drought-responsive genes. Thus, this study provides comprehensive physiological and molecular genetic evidence explaining the lack of wilt symptoms in B. napus infected with V. longisporum. Likewise, drought tolerance of B. napus was unaffected by V. longisporum, as was the level of disease by drought conditions, thus excluding a concerted action of both stresses in the field. Although it is evident that drought and vascular infection with V. longisporum impair plant growth by different mechanisms, it remains to be determined by which other factors V. longisporum causes crop loss.

Wheat Blast and Fusarium Head Blight Display Contrasting Interaction Patterns on Ears of Wheat Genotypes Differing in Resistance.

The interaction of wheat with two ear pathogens, Magnaporthe wheat blast (MWB) and Fusarium graminearum (Fusarium head blight, FHB), was studied on the phenotypic, histological, and gene expression level. Most of the 27 wheat cultivars inoculated with MWB and F. graminearum displayed inverse disease responses to blast and FHB infection. Two cultivars, Milan and Sumai 3, were selected expressing converse disease phenotypes to blast (Milan, R)/(Sumai 3, S) and FHB (Milan, S)/(Sumai 3, R). Confocal laser scanning microscopy revealed early (12 h postinoculation) colonization of the spikelets by MWB similarly on both cultivars, while F. graminearum infected anthers of the susceptible cultivar earlier. Both pathogens grew much faster in the rachilla of susceptible than resistant cultivars, indicating that resistance is mainly expressed in this part connecting the spikelet with the rachis. In general, O2(-) and H2O2 levels were unrelated to disease expression in the four studied interactions. The differential disease phenotypes, fungal spread in the rachis, and colonization patterns in the spikelets were confirmed by distinct gene expression patterns. Among the eight genes analyzed, seven were more strongly induced by FHB than by blast. Genes for chitinase (Chi2), ß-1,3-glucanase (PR2), a plant defensin homolog (PRPI), and peroxidase (Pox2) were strongly upregulated in Milan in response to both pathogens, while PR2 and PR5 (thaumatin-like protein) were transiently triggered by MWB on both cultivars. Upregulation of cinnamoyl-CoA reductase (CCR), cytochrome P450 (CYP709C1), and UDP-glycosyl transferase (UGT) were more prominent in ears infected with F. graminearum, while upregulation of UGT was higher in Sumai 3 when infected with either pathogen. Cultivar resistance to FHB was reflected by clearly higher expression levels of UGT and CYP709C1 in Sumai 3. The differential responses of wheat to the two ear pathogens demonstrated in this study makes it unlikely that common resistance genes exist for control of FHB and blast, suggesting the need to stack many genes associated with resistance in breeding programs for multiple resistance.

Population Structure, Pathogenicity, and Mating Type Distribution of Magnaporthe oryzae Isolates from East Africa.

Rice blast, caused by Magnaporthe oryzae, is one of the emergent threats to rice production in East Africa (EA), where little is known about the population genetics and pathogenicity of this pathogen. We investigated the genetic diversity and mating type (MAT) distribution of 88 isolates of M. oryzae from EA and representative isolates from West Africa (WA) and the Philippines (Asia) using amplified fragment length polymorphism markers and mating-type-specific primer sets. In addition, the aggressiveness of each isolate was evaluated by inoculating on the susceptible Oryza sativa indica 'Co39', scoring the disease severity and calculating the disease progress. Hierarchical analysis of molecular variance revealed a low level of genetic differentiation at two levels (FST 0.12 and FCT 0.11). No evidence of population structure was found among the 65 isolates from EA, and gene flow among EA populations was high. Moreover, pairwise population differentiation (GST) in EA populations ranged from 0.03 to 0.04, suggesting that >96% of genetic variation is derived from within populations. However, the populations from Asia and WA were moderately differentiated from EA ones. The spatial analysis of principal coordinates and STRUCTURE revealed overlapping between individual M. oryzae isolates from EA, with limited distinctness according to the geographic origin. All the populations were clonal, given the positive and significant index of association (IA) and standardized index of association (rd), which indicates a significant (P<0.001) departure from panmixia (IA and rd=0). Both MAT1-1 and MAT1-2 were detected. However, MAT1-1 was more prevalent than MAT1-2. Pathogenicity analysis revealed variability in aggressiveness, suggesting a potential existence of different races. Our data suggest that either M. oryzae populations from EA could be distributed as a single genetic population or gene flow is exerting a significant influence, effectively swamping the action of selection. This is the first study of genetic differentiation of rice-infecting M. oryzae strains from EA, and may guide further studies on the pathogen as well as resistance breeding efforts.

The Three Lineages of the Diploid Hybrid Verticillium longisporum Differ in Virulence and Pathogenicity.

Verticillium longisporum is an economically important vascular pathogen of Brassicaceae crops in different parts of the world. V. longisporum is a diploid hybrid that consists of three different lineages, each of which originated from a separate hybridization event between two different sets of parental species. We used 20 isolates representing the three V. longisporum lineages and the relative V. dahliae, and performed pathogenicity tests on 11 different hosts, including artichoke, cabbage, cauliflower, cotton, eggplant, horseradish, lettuce, linseed, oilseed rape (canola), tomato, and watermelon. V. longisporum was overall more virulent on the Brassicaceae crops than V. dahliae, which was more virulent than V. longisporum across the non-Brassicaceae crops. There were differences in virulence between the three V. longisporum lineages. V. longisporum lineage A1/D1 was the most virulent lineage on oilseed rape, and V. longisporum lineage A1/D2 was the most virulent lineage on cabbage and horseradish. We also found that on the non-Brassicaceae hosts eggplant, tomato, lettuce, and watermelon, V. longisporum was more or equally virulent than V. dahliae. This suggests that V. longisporum may have a wider potential host range than currently appreciated.

Further Limitations of Synthetic Fungicide Use and Expansion of Organic Agriculture in Europe Will Increase the Environmental and Health Risks of Chemical Crop Protection Caused by Copper-Containing Fungicides.

Copper-containing fungicides have been used in agriculture since 1885. The divalent copper ion is a nonbiodegradable multisite inhibitor that has a strictly protective, nonsystemic effect on plants. Copper-containing plant protection products currently approved in Germany contain copper oxychloride, copper hydroxide, and tribasic copper sulfate. Copper is primarily used to control oomycete pathogens in grapevine, hop, potato, and fungal diseases in fruit production. In the environment, copper is highly persistent and toxic to nontarget organisms. The latter applies for terrestric and aquatic organisms such as earthworms, insects, birds, fish, Daphnia, and algae. Hence, copper fungicides are currently classified in the European Union as candidates for substitution. Pertinently, copper also exhibits significant mammalian toxicity (median lethal dose oral = 300-2500 mg/kg body wt in rats). To date, organic production still profoundly relies on the use of copper fungicides. Attempts to reduce doses of copper applications and the search for copper substitutes have not been successful. Copper compounds compared with modern synthetic fungicides with similar areas of use display significantly higher risks for honey bees (3- to 20-fold), beneficial insects (6- to 2000-fold), birds (2- to 13-fold), and mammals (up to 17-fold). These data contradict current views that crop protection in organic farming is associated with lower environmental or health risks. Further limitations in the range and use of modern single-site fungicides may force conventional production to fill the gaps with copper fungicides to counteract fungicide resistance. In contrast to the European Union Green Deal goals, the intended expansion of organic farming in Europe would further enhance the use of copper fungicides and hence increase the overall risks of chemical crop protection in Europe. Environ Toxicol Chem 2024;43:19-30. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Mechanisms regulating grain contamination with trichothecenes translocated from the stem base of wheat (Triticum aestivum) infected with Fusarium culmorum.

Factors limiting trichothecene contamination of mature wheat grains after Fusarium infection are of major interest in crop production. In addition to ear infection, systemic translocation of deoxynivalenol (DON) may contribute to mycotoxin levels in grains after stem base infection with toxigenic Fusarium spp. However, the exact and potential mechanisms regulating DON translocation into wheat grains from the plant base are still unknown. We analyzed two wheat cultivars differing in susceptibility to Fusarium head blight (FHB), which were infected at the stem base with Fusarium culmorum in climate chamber experiments. Fungal DNA was found only in the infected stem base tissue, whereas DON and its derivative, DON-3-glucoside (D3G), were detected in upper plant parts. Although infected stem bases contained more than 10,000 µg kg⁻¹ dry weight (DW) of DON and mean levels of DON after translocation in the ear and husks reached 1,900 µg kg⁻¹ DW, no DON or D3G was detectable in mature grains. D3G quantification revealed that DON detoxification took mainly place in the stem basis, where ≤ 50% of DON was metabolized into D3G. Enhanced expression of a gene putatively encoding a uridine diphosphate-glycosyltransferase (GenBank accession number FG985273) was observed in the stem base after infection with F. culmorum. Resistance to F. culmorum stem base infection, DON glycosylation in the stem base, and mycotoxin translocation were unrelated to cultivar resistance to FHB. Histological studies demonstrated that the vascular transport of DON labeled with fluorescein as a tracer from the peduncle to the grain was interrupted by a barrier zone at the interface between grain and rachilla, formerly described as "xylem discontinuity". This is the first study to demonstrate the effective control of influx of systemically translocated fungal mycotoxins into grains at the rachilla-seed interface by the xylem discontinuity tissue in wheat ears.

Interactions between the predatory mite Typhlodromalus aripo and the entomopathogenic fungus Neozygites tanajoae and consequences for the suppression of their shared prey/host Mononychellus tanajoa.

The predatory mite Typhlodromalus aripo and the entomopathogenic fungus Neozygites tanajoae, both introduced from Brazil for control of the cassava green mite (CGM) Mononychellus tanajoa, now co-occur in cassava fields in Benin. However, studies on interactions between these two natural enemies and how they might affect CGM biological control are lacking. We determined in screenhouse experiments the effects of single and combined releases of N. tanajoae and T. aripo on CGM suppression. In the single natural enemy treatment, both T. aripo and N. tanajoae significantly reduced CGM densities, but the results of the predator (T. aripo) are more quickly measurable than those of the pathogen (N. tanajoae) in our short-term experiment. The level of CGM suppression in the combined natural enemy treatment was reduced considerably compared with T. aripo-alone, but only slightly when compared with N. tanajoae alone, with a simultaneous reduction in T. aripo and N. tanajoae abundance or prevalence. In a laboratory experiment, T. aripo fed more on N. tanajoae-infected CGM than on healthy CGM and its oviposition and survival were reduced when fed on the former compared with the latter, which can help in explaining the reduction in numbers of T. aripo and consequently the considerable loss in suppression of CGM in the combined natural enemy treatment in the screenhouse experiment. Together, the screenhouse and the laboratory experiments predicted negative interactions between the two natural enemies with negative consequences for CGM biological control. Long-term field observations and rigorous field experiments that simultaneously manipulate T. aripo and N. tanajoae abundance and prevalence are needed to validate the prediction of this study.

Pathogenicity of Trichoderma afroharzianum in Cereal Crops.

Species of the genus Trichoderma occur ubiquitously in soils, on plant roots and in decaying plant residues. Due to its competitiveness and mycoparasitic potential against other microorganisms, particular strains of Trichoderma spp. are used in agriculture as biocontrol agents against plant pathogens. However, Trichoderma afroharzianum has been recently reported as a pathogen causing ear rot disease on maize in Germany, France and Italy, leading to massive infections on maize cobs. This raised the question, whether and to what extent Trichoderma spp. can infect cereal crops other than maize and cause disease symptoms and yield losses. To address this question, two varieties of wheat, barley and sorghum were grown in the greenhouse and artificially inoculated with T. afroharzianum by both spray and point inoculation at the time of flowering. Disease severity was scored weekly, and thousand-kernel weight and colonization rate were determined after harvest. As early as 14 days after inoculation, the first visual symptoms appeared on wheat and barley as tan or brown discoloration of the base of a floret within the spikelets. After spray inoculation, clear discolorations of the entire ear were seen, while point inoculation only showed symptoms at the injection site and above. No visible symptoms were observed on sorghum millet. The colonization rate on wheat and barley grains was significantly increased compared to the control, while thousand-kernel weights (TKWs) were significantly reduced. No differences in colonization rate and TKW compared to the control were observed in sorghum. This is the first report of Trichoderma afroharzianum infecting wheat and barley, causing disease symptoms and significantly reducing thousand-kernel weights.

Role of seed infection for the near and far distance dissemination of wheat blast caused by Magnaporthe oryzae pathotype Triticum.

Magnaporthe oryzae pathotype Triticum (MoT) is a devastating fungal phytopathogen causing wheat blast disease which threatens wheat production particularly in warmer climate zones. Effective disease control is hampered by the limited knowledge on the life cycle, epidemiology, and pathogenicity of MoT. Since MoT mainly infects and colonizes the inflorescences of wheat, infection, invasion routes and colonization of MoT on wheat ears and in wheat seeds were investigated in order to assess potential seed transmission pathways. MoT was spray inoculated on two wheat cultivars (Sumai 3, susceptible and Milan, resistant) at three ear maturity stages [full ear emergence, growth stage (GS) 59; mid flowering, GS 65; and end of flowering, GS 69]. Incidence of MoT on Sumai 3 seeds was 100% and 20-25% on Milan. MoT sporulation rate on Sumai 3 contaminated seeds was more than 15 times higher than on Milan. Repeated washes of seed samples for removing paraffin fixation hampers seed microscopy. To overcome the damage of seed samples, we used hand-sectioned seed samples instead of paraffin-fixed microtome samples to facilitate microscopy. The colonization of MoT within various seed tissues was followed by light and confocal laser scanning microscopy (CLSM). Invasion of MoT in seeds predominantly occurred in the caryopsis germ region, but entry via other seed parts was also observed, confirming the potential of intense colonization of MoT in wheat grains. Fungal spread in wheat plants growing from MoT infected seeds was monitored through plating, microscopic and molecular techniques. Under greenhouse conditions, no spread of MoT from infected seeds to seedlings later than GS 21 or to ears was detected, neither in Milan nor in Sumai 3. We therefore conclude, that MoT may not systemically contaminate inflorescences and seeds in neither susceptible nor resistant wheat cultivars. However, initial blast symptoms, only found on seedlings of Sumai 3 but not Milan, resulted in the formation of new conidia, which may serve as inoculum source for plant-to-plant dissemination by airborne infection of plant stands in the field (short distance spread). Ultimately the inoculum may infect young inflorescences in the field and contaminate seeds. Our findings again stress the risk of long-distance dissemination of wheat blast across continents through MoT-contaminated seeds. This underlines the importance of mandatory use of healthy seeds in strategies to control any further spread of wheat blast.

Population Genomic Evidence for a Repeated Introduction and Rapid Expansion of the Fungal Maize Pathogen Setosphaeria turcica in Europe.

Modern agricultural practices, climate change, and globalization foster the rapid spread of plant pathogens, such as the maize fungal pathogen Setosphaeria turcica, which causes Northern corn leaf blight and expanded into Central Europe during the twentieth century. To investigate the rapid expansion of S. turcica, we sequenced 121 isolates from Europe and Kenya. Population genomic inference revealed a single genetically diverse cluster in Kenya and three clonal lineages with low diversity, as well as one cluster of multiple clonal sublineages in Europe. Phylogenetic dating suggests that all European lineages originated through sexual reproduction outside Europe and were subsequently introgressed multiple times. Unlike isolates from Kenya, European isolates did not show sexual recombination, despite the presence of both MAT1-1 and MAT1-2 mating types. For the clonal lineages, coalescent model selection supported a selectively neutral model with strong exponential population growth, rather than models with pervasive positive selection caused by host defense resistance or environmental adaptation. Within clonal lineages, phenotypic variation in virulence to different monogenic resistances, which defines the pathogen races, suggests that these races may originate from repeated mutations in virulence genes. Association testing based on k-mers did not identify genomic regions linked to pathogen races, but it did uncover strongly differentiated genomic regions between clonal lineages, which harbor genes with putative roles in pathogenicity. In conclusion, the expansion and population growth of S. turcica in Europe are mainly driven by an expansion of the maize cultivation area and not by rapid adaptation.

Suppressive Effects of Volatile Compounds from Bacillus spp. on Magnaporthe oryzae Triticum (MoT) Pathotype, Causal Agent of Wheat Blast.

The Magnaporthe oryzae Triticum (MoT) pathotype is the causal agent of wheat blast, which has caused significant economic losses and threatens wheat production in South America, Asia, and Africa. Three bacterial strains from rice and wheat seeds (B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) were used to explore the antifungal effects of volatile organic compounds (VOCs) of Bacillus spp. as a potential biocontrol mechanism against MoT. All bacterial treatments significantly inhibited both the mycelial growth and sporulation of MoT in vitro. We found that this inhibition was caused by Bacillus VOCs in a dose-dependent manner. In addition, biocontrol assays using detached wheat leaves infected with MoT showed reduced leaf lesions and sporulation compared to the untreated control. VOCs from B. velezensis BTS-4 alone or a consortium (mixture of B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) of treatments consistently suppressed MoT in vitro and in vivo. Compared to the untreated control, VOCs from BTS-4 and the Bacillus consortium reduced MoT lesions in vivo by 85% and 81.25%, respectively. A total of thirty-nine VOCs (from nine different VOC groups) from four Bacillus treatments were identified by gas chromatography-mass spectrometry (GC-MS), of which 11 were produced in all Bacillus treatments. Alcohols, fatty acids, ketones, aldehydes, and S-containing compounds were detected in all four bacterial treatments. In vitro assays using pure VOCs revealed that hexanoic acid, 2-methylbutanoic acid, and phenylethyl alcohol are potential VOCs emitted by Bacillus spp. that are suppressive for MoT. The minimum inhibitory concentrations for MoT sporulation were 250 mM for phenylethyl alcohol and 500 mM for 2-methylbutanoic acid and hexanoic acid. Therefore, our results indicate that VOCs from Bacillus spp. are effective compounds to suppress the growth and sporulation of MoT. Understanding the MoT sporulation reduction mechanisms exerted by Bacillus VOCs may provide novel options to manage the further spread of wheat blast by spores.