Baseline sensitivity and control efficacy of fluazinam against Sclerotinia sclerotiorum in Henan Province,China

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a devastating disease in Henan Province, of the main rapeseed production areas in China. Fluazinam belongs to the broad‐spectrum phenylpyridinamine fungicides, which have high activity in inhibiting the mycelial growth of S. sclerotiorum. In this study, 191 field isolates were obtained from different oilseed rape fields in Henan Province, before being exposed to fluazinam in 2015. The baseline sensitivity of S. sclerotiorum to fluazinam was established. The effective concentration for 50% inhibition of mycelial growth (EC₅₀) ranged from 0.0019 to 0.0337 μg/ml, and the mean EC₅₀ value was 0.0084 ± 0.0055 μg/ml. The range of the frequency distribution was narrow. The results of a cross‐resistance assay revealed no cross‐resistance between fluazinam and carbendazim, dimethachlone, boscalid or fludioxonil. Field efficacy tests showed that the control efficacies of fluazinam (50% WG) applied at 150, 225 and 300 g ai ha^?1 were 67%, 73% and 88%, respectively. In contrast, the control efficacies of boscalid (50% WG) and carbendazim (50% WP) applied at 225 and 1,500 g ai ha^?1 were 71% and 52%, respectively.     

Sensitivity to boscalid in field isolates of Sclerotinia sclerotiorum from rapeseed in Henan Province,China

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is an important disease of oilseed rape in Henan province of China. Boscalid belongs to succinate dehydrogenase inhibitor (SDHI) fungicides, many of which have strong antifungal activity against S. sclerotiorum. In 2015, a total of 175 isolates of S. sclerotiorum were collected from diseased oilseed rape plants in seven different regions of Henan Province. The EC₅₀ values of 175 isolates of S. sclerotiorum to boscalid ranged from 0.0073 to 0.3880 μg ml^?1, and the mean EC₅₀ value was 0.15 ± 0.09 μg ml^?1. The frequency distribution was unimodal. There was no cross‐resistance between boscalid and carbendazim, procymidone, iprodione, dimethachlone, fludioxonil or fluazinam. Field experiments showed that control efficacies of treatments with boscalid (50% WG) at 225, 300 and 375 g ai ha^?1 were 71%, 81% and 90%, respectively. In contrast, the control efficacy of carbendazim (50% WP) at 1,500 g ai ha^?1 was only 52%.     

A new point mutation in the iron–sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum

Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid‐resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid‐sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild‐type strains, BR and GSM (SdhB gene in the wild‐type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron–sulfur subunit of SDH. These results suggest that resistance based on non‐conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs.     

Baseline sensitivities to azoxystrobin and tebuconazole in Sclerotinia sclerotiorum isolates from sunflower in Iran related to sensitivities to carbendazim and iprodione

In this study, sensitivities of 156 Sclerotinia sclerotiorum isolates collected from sunflower fields of West Azarbaijan province, Iran, were assessed to carbendazim and iprodione, and the baseline sensitivities were established for azoxystrobin and tebuconazole. Resistance to carbendazim and iprodione was observed in 53.85% and 4.49% of the isolates, respectively. The 50% effective concentration (EC₅₀) values of azoxystrobin for the isolates ranged from 0.017 to 3.515 μg/ml with a mean of 0.330 μg/ml, and 8.97% of the strains showed low levels of resistance to the fungicide. However, in the presence of salicylhydroxamic acid, all isolates were sensitive to azoxystrobin and EC₅₀ values ranged from 0.015 to 0.263 μg/ml with a mean of 0.086 μg/ml. All isolates were found to be sensitive to tebuconazole, and EC₅₀ values ranged from 0.003 to 0.177 μg/ml with a mean of 0.036 μg/ml. Among the multiple-resistant isolates, the strains exhibiting resistance to both carbendazim and iprodione were detected in the highest frequency (4.49%). No correlation was observed between mycelial growth and aggressiveness with fungicide sensitivity of the isolates suggesting the absence of fitness cost associated with resistance to the studied fungicides. The results indicated that iprodione, azoxystrobin and tebuconazole could be effectively used in rotation or mixture in spray programmes to manage S. sclerotiorum in the region. The baselines established for azoxystrobin and tebuconazole would be useful in monitoring the fungal populations in the province to assess possible shifts in fungicide sensitivity of S. sclerotiorum isolates in the future.     

Rapid identification of an Arabidopsis NLR gene as a candidate conferring susceptibility to Sclerotinia sclerotiorum using time‐resolved automated phenotyping

The broad host range necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen of many oil and vegetable crops. Plant genes conferring complete resistance against S. sclerotiorum have not been reported. Instead, plant populations challenged by S. sclerotiorum exhibit a continuum of partial resistance designated as quantitative disease resistance (QDR). Because of their complex interplay and their small phenotypic effect, the functional characterization of QDR genes remains limited. How broad host range necrotrophic fungi manipulate plant programmed cell death is for instance largely unknown. Here, we designed a time‐resolved automated disease phenotyping pipeline enabling high‐throughput disease lesion measurement with high resolution, low footprint at low cost. We could accurately recover contrasted disease responses in several pathosystems using this system. We used our phenotyping pipeline to assess the kinetics of disease symptoms caused by seven S. sclerotiorum isolates on six A. thaliana natural accessions with unprecedented resolution. Large effect polymorphisms common to the most resistant A. thaliana accessions identified highly divergent alleles of the nucleotide‐binding site leucine‐rich repeat gene LAZ5 in the resistant accessions Rubezhnoe and Lip‐0. We show that impaired LAZ5 expression in laz5.1 mutant lines and in A. thaliana Rub natural accession correlate with enhanced QDR to S. sclerotiorum. These findings illustrate the value of time‐resolved image‐based phenotyping for unravelling the genetic bases of complex traits such as QDR. Our results suggest that S. sclerotiorum manipulates plant sphingolipid pathways guarded by LAZ5 to trigger programmed cell death and cause disease.     

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent

Aims: To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent. Methods and Results: The antagonistic activity of C. rosea BAFC3874 was determined in vitro by dual cultures. The strain effectively antagonized S. sclerotiorum in pot‐grown lettuce and soybean plants. Antifungal activity assays of C. rosea BAFC3874 grown in culture established that the strain produced antifungal compounds against S. sclerotiorum associated with secondary metabolism. High mycelial growth inhibition coincided with sclerotia production inhibition. The C. rosea strain produced a microheterogeneous mixture of peptides belonging to the peptaibiotic family. Moreover, mycoparasitism activity was observed in the dual culture. Conclusions: Clonostachys rosea strain BAFC3874 was proved to be an effective antagonist against the aggressive soil‐borne pathogen S. sclerotiorum in greenhouse experiments. The main mechanisms involve peptaibiotic metabolite production and mycoparasitism activity. Significance and Impact of the Study: Clonostachys rosea BAFC3874 may be a good fungal biological control agent against S. sclerotiorum. In addition, we were also able to isolate and identify peptaibols, an unusual family of compounds in this genus of fungi.     

Assessing in vitro efficacy of certain fungicides to control Sclerotinia sclerotiorum in peanut

The fungal pathogen Sclerotinia sclerotiorum is responsible for Sclerotinia blight in several crops around the world, including peanut. This study was conducted under laboratory conditions to determine the effects of four registered fungicides, Propulse?, Fontelis^®, Omega^® and Endura^® on mycelial growth and pigmentation, as well as sclerotia and oxalic acid production on a growth medium modified with a fungicide and on the pathogenicity of S. sclerotiorum on leaflets detached from Valencia peanut. Propulse, Omega and Fontelis inhibited mycelial growth of S. sclerotiorum, while, mycelial growth on a modified support with Endura was similar to the control treatment. All fungicides, except Endura, inhibited the production of oxalic acid. Pigmentation of the mycelium was observed in both the control and endura treatments. Sclerotia production was observed only in the control treatment. With the exception of Endura, all fungicides were effective in controlling the development of lesions on Valencia peanut leaflets.     

Molecular genotyping of Sclerotinia sclerotiorum isolates from different regions and host plants in Iran

Genetic variation among Sclerotinia sclerotiorum isolates from different regions and host plants were investigated using pathogenicity test, mycelial compatibility groups (MCGs) and molecular markers. Six MCGs were identified and significant differences of virulence variability were observed within and among MCGs. Cluster analysis of combined repetitive sequence-based polymerase chain reaction and randomly amplified polymorphic DNA data discriminated 12 isolates into 11 genotypes, indicating high level of genetic polymorphism among tested isolates. Twelve isolates clustered into four major groups corresponding to their hosts andgeographical region. The variability found within closely related isolates of S.sclerotiorum indicated that such morphological and molecular markers are useful in population studies of this pathogen.     

Morphological and Pathogenic Characterization of Genetically Diverse Sclerotinia Isolates from European Red Clover Crops (Trifolium Pratense L.)

Clover rot, an important disease in European red clover crops, is caused by Sclerotinia trifoliorum or Sclerotinia sclerotiorum. Until today, little is known about the variation in aggressiveness among Sclerotinia isolates from red clover. Aggressiveness has never been correlated with morphological characteristics. Rapidly growing isolates may be more aggressive, but this was never investigated in S. trifoliorum before. Also nothing is known about the link between sclerotia production and aggressiveness. Oxalic acid is an important pathogenicity factor in Sclerotinia species, but its effect on aggressiveness is unknown in S. trifoliorum isolates. For this study, we selected 30 Sclerotinia isolates from 25 locations Europe: 26 S. trifoliorum isolates and 4 S. sclerotiorum isolates from two locations in France (Fr.A and Fr.B). For each isolate, the in vitro growth speed, sclerotia production, oxalate production and aggressiveness were analysed and correlations were estimated between aggressiveness and the other characteristics. Aggressiveness was assessed in vitro on detached leaves and in a greenhouse on young plants. Our isolates differed significantly in growth speed, sclerotia production, oxalate production and aggressiveness. The infections on detached leaves and young plants revealed interaction between isolates and plant genotypes and between isolates and cultivars, but there was no indication that pathotypes exist. In vitro growth speed and in vitro aggressiveness on detached leaves were positively correlated with aggressiveness on young plants, while sclerotia production was negatively correlated with aggressiveness on young plants. These factors can be used as predictors of aggressiveness of Sclerotinia isolates from red clover crops.     

Suppression of carbendazim-susceptible and -resistant Sclerotinia sclerotiorum with Macleya alkaloids

Macleaya alkaloids (abr. MCA), an extract from aerial parts of Macleaya cordata, was investigated on suppressing Sclerotinia stem rot disease. The median inhibitory concentrations (EC₅₀) of MCA on mycelia growth were 5.21 μg mL^?1 to carbendazim-susceptible (Ss01) and 6.34 μg mL^?1 to carbendazim-resistant (Hm25) S. sclerotiorum, and there was no cross-resistance between MCA and carbendazim. MCA blocked the mycelial membrane leakage and regulated the exo-secretion of the reducing sugar and oxalate in a concentration-dependent manner. Moreover, MCA also significantly destroyed the redox balance including superoxide dismutase, peroxidase and catalase in Sclerotinia mycelia. In pot experiments, MCA showed an excellent antifungal efficacy on protecting rapeseed leaves from the infection of Ss01 and Hm25 isolates. The results suggested a potential possibility to develop MCA as an agro-chemical to control Sclerotinia stem rot disease and manage carbendazim resistance.     

An atypical forkhead‐containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)‐box‐containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)‐based gene silencing was employed to alter the expression of SsFkh1. RNA‐silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis‐related laccase genes and a polyketide synthase‐encoding gene were significantly down‐regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi‐silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.     

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of M?ule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.     

Contribution of Pathogens to Peach Fruit Rot in Northern Greece and their Sensitivity to Iprodione, Carbendazim, Thiophanate-methyl and Tebuconazole Fungicides

This study identified the main pathogens causing fruit rots of mature peaches in northern Greece, the major peach producing area of Greece. The brown rot pathogen Monilinia laxa was responsible for approximately 70% and 78% of rotted peaches in 2005 and 2006 respectively. Serious damage (up to 5%) was also caused with the fungus Phomopsis amygdali. Other pathogens isolated from rotted peaches at a low percentage were Alternaria alternata, Aspergillus niger, Aspergillus flavus, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium spp., Colletotrichum gloeosporioides, Rhizopus stolonifer and Gilbertella persicaria. Most fungal isolates originated from the rotted peaches were tested for their sensitivity to the fungicides iprodione, carbendazim, thiophanate methyl and tebuconazole at label recommended concentrations. All fungicides inhibited the growth of M. laxa, A. niger, A. flavus, S. sclerotiorum, P. amygdali and B. cinerea on poisoned agar. Apart from iprodione, all other fungicides inhibited the mycelium growth of the pathogen Fusarium sp. The mycelium growth of Fusarium sp. was significantly less with iprodione than control. Only iprodione and tebuconazole were effective against A. alternata and R. stolonifer. Tebuconazole inhibited the mycelium growth of R. stolonifer, while iprodione reduced significantly in comparison to control. The mycelium growth of the fungus C. gloeosporioides was inhibited by tebuconazole and reduced significantly by the fungicides thiophanate methyl, carbendazim and iprodione. Among all the fungi tested, only M. laxa and B. cinerea isolates were found resistant to benzimidazoles [the EC50 (50% effective concentration) value was 100–200 mg/l and 200–300 mg/l for the largest number of thiophanate methyl‐ and carbendazim‐resistant M. laxa isolates respectively, while the biggest number of B. cinerea thiophanate methyl‐ and carbendazim‐resistant isolates showed EC50 value 200–300 mg/l and 300–400 mg/l, respectively]. However, these strains were sensitive to tebuconazole and iprodione. Therefore, these fungicides can be used as an alternative method to control benzimidazole‐resistant Monilinia and Botrytis isolates.     

Valuable New Resistances Ensure Improved Management of Sclerotinia Stem Rot (Sclerotinia sclerotiorum) in Horticultural and Oilseed Brassica Species

Field resistances against Sclerotinia rot (SR) (Sclerotinia sclerotiorum) were determined in 52 Chinese genotypes of Brassica oleracea var. capitata, 14 Indian Brassica juncea genotypes carrying wild weedy Brassicaceae introgression(s) and four carrying B‐genome introgression, 22 Australian commercial Brassica napus varieties, and 12 B. napus and B. juncea genotypes of known resistance. All plants were individually inoculated by securing an agar disc from a culture of S. sclerotiorum growing on a glucose‐rich medium to the stem above the second internode with Parafilm tape. Mean stem lesion length across tested genotypes ranged from <1 to >68 mm. While there was considerable diversity within the germplasm sets from each country, overall, 65% of the B. oleracea var. capitata genotypes from China showed the highest levels of stem resistance, a level comparable with the highest resistance ever recorded for oilseed B. napus or B. juncea from China or Australia. One Indian B. juncea line carrying weedy introgression displayed a significant level of both stem and leaf resistance. However, the vast majority of commercial Australian oilseed B. napus varieties fell within the most susceptible 40% of genotypes tested for stem disease. There was no correlation between expressions of stem versus leaf resistance, suggesting their independent inheritance. A few Chinese B. oleracea var. capitata genotypes that expressed combined extremely high‐level stem (≤1 mm length) and leaf (≤0.5 mean number of infections/plant) resistance will be particularly significant for developing new SR‐resistant horticultural and oilseed Brassica varieties.