The velvet family proteins mediate low resistance to isoprothiolane in Magnaporthe oryzae.

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 µg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 µg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 µg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT.

Bioproducts of Pseudomonas chlororaphis Suppress DMI Fungicide-Induced CsCYP51A and CsCYP51B Gene Expression in Colletotrichum siamense and Generate Synergistic Effects with Metconazole and Propiconazole.

Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below-label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry (Prunus avium) caused by Colletotrichum siamense. Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Real-time PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense. The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays.

Fungicide resistance in Colletotrichum fructicola and Colletotrichum siamense causing peach anthracnose in China.

Peach is one of the popular and economically important fruit crops in China. Peach cultivation is hampered due to attacks of anthracnose disease, causing significant economic losses. Colletotrichum fructicola and Colletotrichum siamense belong to the Colletotrichum gloeosporioides species complex and are considered major pathogens of peach anthracnose. Application of different groups of fungicides is a routine approach for controlling this disease. However, fungicide resistance is a significant drawback in managing peach anthracnose nowadays. In this study, 39 isolates of C. fructicola and 41 isolates of C. siamense were collected from different locations in various provinces in China. The sensitivity of C. fructicola and C. siamense to some commonly used fungicides, i.e., carbendazim, iprodione, fluopyram, and propiconazole, was determined. All the isolates of C. fructicola collected from Guangdong province showed high resistance to carbendazim, whereas isolates collected from Guizhou province were sensitive. In C. siamense, isolates collected from Hebei province showed moderate resistance, while those from Shandong province were sensitive to carbendazim. On the other hand, all the isolates of C. fructicola and C. siamense showed high resistance to the dicarboximide (DCF) fungicide iprodione and succinate dehydrogenase inhibitor (SDHI) fungicide fluopyram. However, they are all sensitive to the demethylation inhibitor (DMI) fungicide propiconazole. Positive cross-resistance was observed between carbendazim and benomyl as they are members of the same methyl benzimidazole carbamate (MBC) group. While no correlation of sensitivity was observed between different groups of fungicides. No significant differences were found in each fitness parameter between carbendazim-resistant and sensitive isolates in both species. Molecular characterization of the ß-tubulin 2 (TUB2) gene revealed that in C. fructicola, the E198A point mutation was the determinant for the high resistance to carbendazim, while the F200Y point mutation was linked with the moderate resistance to carbendazim in C. siamense. Based on the results of this study, DMI fungicides, e.g., propiconazole or prochloraz could be used to control peach anthracnose, especially at locations where the pathogens have already developed the resistance to carbendazim and other fungicides.

Pseudomonas chlororaphis Metabolites Reduce MfCYP51 Expression and Yield Synergistic Efficacy in Mixture with Reduced Rates of Propiconazole Against DMI-Resistant Monilinia fructicola Isolates.

Brown rot caused by Monilinia fructicola is one of the most important diseases affecting peach production in the southeastern United States. Management often involves the use of demethylation inhibitor (DMI) fungicides, but efficacy can be compromised because of overexpression of the MfCYP51 gene encoding the 14α-demethylase of the ergosterol biosynthesis pathway. This study aimed to investigate the influence of the biorational fungicide Howler EVO containing Pseudomonas chlororaphis ASF009 metabolites on the expression of MfCYP51 in M. fructicola and associated synergy with a DMI fungicide for control of DMI-resistant strains. Mycelia from two DMI-sensitive and three DMI-resistant M. fructicola isolates were exposed or not to propiconazole (0.3 µg/ml), Howler (88.1 µg/ml), or the combination propiconazole + Howler for 6 h prior to RNA extraction. Real-time PCR indicated that Howler reduced the constitutive expression of MfCYP51 in DMI-sensitive and two of three DMI-resistant isolates. Propiconazole-induced expression of the DMI target gene was significantly reduced by Howler and by the mixture of Howler plus propiconazole in all isolates. Detached fruit studies on apple revealed that the combination of Howler plus a reduced label rate of Mentor (50 µg/ml propiconazole) was synergistic against brown rot caused by a DMI-resistant isolate in high and low inoculum spore concentration experiments (synergy values of 40.1 and 4.9, respectively). We hypothesize that the synergistic effects against M. fructicola resistant to DMI fungicides based on MfCYP51 gene overexpression can be attributed to reduced 14α demethylase production due to transcription inhibition, which may necessitate fewer DMI fungicide molecules to arrest fungal growth. The use of Howler/DMI mixtures for brown rot control warrants further investigation because such mixtures could potentially allow for reduced DMI fungicide use rates in the field without compromising yield or increased resistance selection.

Melatonin targets MoIcl1 and works synergistically with fungicide isoprothiolane in rice blast control.

Melatonina natural harmless molecule-displays versatile roles in human health and crop disease control such as for rice blast. Rice blast, caused by the filamentous fungus Magnaporthe oryzae, is one devastating disease of rice. Application of fungicides is one of the major measures in the control of various crop diseases. However, fungicide resistance in the pathogen and relevant environmental pollution are becoming serious problems. By screening for possible synergistic combinations, here, we discovered an eco-friendly combination for rice blast control, melatonin, and the fungicide isoprothiolane. These compounds together exhibited significant synergistic inhibitory effects on vegetative growth, conidial germination, appressorium formation, penetration, and plant infection by M. oryzae. The combination of melatonin and isoprothiolane reduced the effective concentration of isoprothiolane by over 10-fold as well as residual levels of isoprothiolane. Transcriptomics and lipidomics revealed that melatonin and isoprothiolane synergistically interfered with lipid metabolism by regulating many common targets, including the predicted isocitrate lyase-encoding gene MoICL1. Furthermore, using different techniques, we show that melatonin and isoprothiolane interact with MoIcl1. This study demonstrates that melatonin and isoprothiolane function synergistically and can be used to reduce the dosage and residual level of isoprothiolane, potentially contributing to the environment-friendly and sustainable control of crop diseases.

Site-directed transformants with E407K substitution in Bcmdl1 possesses different fitness from field anilinopyrimidine resistant isolates with E407K mutation in Botrytis cinerea.

Botrytis cinerea is the causal agent of devastating disease gray mold on numerous crops worldwide. To control gray mold, anilinopyrimidine (AP) fungicides have been widely applied since the 1990s. However, the development of resistance in B. cinerea brought a new challenge to this disease control. Due to the unknown mode of action, the mechanism of AP resistance is still ambiguous. In our previous study, mutation E407K in Bcmdl1 was identified to be associated with AP resistance. Since this mutation is the major mechanism of AP resistance in our cases, it is essential to investigate the fitness of E407K strains before designing anti-resistance management strategies. Besides using field-resistant isolates with the E407K mutation, strains with E407K substitution obtained by site-directed mutagenesis were also used to estimate the specific effect of this mutation or substitution on fitness. The fitness of E407K strains were evaluated by determining mycelial growth, sporulation, conidial germination, virulence, acid production, osmotic and oxidative sensitivity, and sclerotial production and viability. Field resistant isolates with E407K mutation produced fewer sclerotia on intermediate medium (IM) but more conidia on PDA when compared with sensitive isolates, whereas site-directed transformants with E407K substitution did not show any fitness costs. The competitive ability of E407K strains was also evaluated on apple fruit using conidial mixtures at three initial ratios of resistant and sensitive isolates at 1:9, 1:1, and 9:1, respectively. Similar with fitness, impaired competitive ability was observed in field resistant isolates but not site-directed transformants at all initial ratios tested. These results indicated that field strains associated with AP resistance suffer a fitness penalty not linked directly to the E407K substitution in Bcmdl1.

Gap-Free Nuclear and Mitochondrial Genomes of Ustilaginoidea virens JS60-2, a Fungal Pathogen Causing Rice False Smut.

Rice false smut (RFS), caused by Ustilaginoidea virens, has become a major disease in recent years, and mycotoxins produced by U. virens often threaten food safety. To study fungal pathogenesis and identify potential targets for developing new fungicides, gap-free nuclear and complete mitochondrial genomes of U. virens JS60-2 were sequenced and assembled. Using the second and third generation sequencing data, we assembled a 38.02-Mb genome that consists of seven contigs with the contig N50 being 6.32-Mb. In total, 8,486 protein-coding genes were annotated in the genome, including 21 secondary metabolism gene clusters. We also assembled the complete mitochondrial genome, which is 102,498 bp, with 28% GC content. The JS60-2 genomes assembled in this study will facilitate research on U. virens and contribute to RFS control. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Benzimidazole-Resistant Isolates with E198A/V/K Mutations in the ß-Tubulin Gene Possess Different Fitness and Competitive Ability in Botrytis cinerea.

Previous studies in Botrytis cinerea showed that resistance to methyl benzimidazole carbamates (MBCs) was mainly related to E198A/V/K and F200Y mutations of the ß-tubulin gene, and E198V was the dominant mutation in the resistant subpopulation in Hubei Province of China, indicating that resistant mutations might influence fitness. However, little is known about the effect of each E198A/V/K mutation on fitness. In this study, the fitness and competitive ability of isolates with E198A/V/K mutations were investigated. Results showed that E198A/V/K isolates and wild-type isolates shared similar fitness components in terms of virulence, sporulation, conidial germination, oxidative sensitivity, and sclerotial production and viability. However, slower mycelial growth at 4°C, higher sensitivity to 4% NaCl, and increased sclerotial production percentage at 4°C were observed in the isolates with E198V, E198K, and E198A mutations, respectively. Competitive analysis showed that the wild-type subpopulation became dominant after three disease cycles in the absence of fungicide selection pressure, whereas the resistant subpopulation seized the space of the sensitive subpopulation upon MBC application. Unexpectedly, the frequency of E198V isolates decreased dramatically after the first disease cycle with or without fungicide selection pressure. These results suggest that MBC-resistant isolates suffer little fitness penalty but possess competitive disadvantages in the absence of fungicide selection pressure. Under fungicide selection pressure, E198V isolates could not compete with E198A/K isolates. According to the current results, there is a great possibility that the E198V mutation will lose dominance in the future in China.

Identification of Monilia Species in Tibet and Characterization of M. yunnanensis in China.

Samples of peach and plum fruits with brown rot symptoms were collected from Tibet in 2019 and 2020, and the causal agent was identified as Monilia yunnanensis, which represents the first characterization of Monilia spp. on peach and plum in Tibet. Morphological investigation showed that some conidia from naturally diseased fruits were larger than those observed in previously isolated M. yunnanensis. Some conidia of M. yunnanensis isolates from Tibet produced more than two, even up to six germ tubes from different parts of each conidium, instead of one or two germ tubes developing from the pointy sides of each conidium. The alignment of ribosomal internal transcribed spacer region sequences revealed that some isolates from Tibet displayed a mutation at the 374th position from adenine (A) to cytosine (C). Although abovementioned differences were observed between isolates from Tibet and other regions, phylogenetic analysis indicated that all of the M. yunnanensis isolates from different stone fruits and different regions in China were clustered together without obvious genetic differentiation. These results revealed that hosts and geographic environments did not play a major role in the evolution of M. yunnanensis.

Cytological Observation of the Infectious Process of Venturia carpophila on Peach Leaves.

Peach scab caused by Venturia carpophila is one of the most destructive fungal diseases of peach worldwide, and it seriously affects peach production. Until now,the infectious process and pathogenesis of V. carpophila on peach have remained unclear. Here we present the infection behavior of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (i.e., light microscopy, confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer and then differentiated into thick subcuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the subcuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the subcuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the subcuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further a understanding of V. carpophila pathogenesis.

Sensitivity of Colletotrichum nymphaeae to Six Fungicides and Characterization of Fludioxonil-Resistant Isolates in China.

Colletotrichum nymphaeae is the dominant species causing anthracnose disease of peach in China. In this study, 140 isolates of C. nymphaeae were assessed for their sensitivity to six fungicides. It was found that C. nymphaeae was highly resistant to carbendazim, procymidone, and boscalid but sensitive to pyraclostrobin and prochloraz. For fludioxonil, the fungus exhibited differential sensitivities (i.e., approximately 14% of isolates were resistant to fludioxonil and the resistance was stable). Fludioxonil-resistant isolates had a mean EC50 value of 2.2380 µg/ml, whereas the mean EC50 value was 0.0194 µg/ml in fludioxonil-sensitive isolates. The mean EC50 values of C. nymphaeae for pyraclostrobin and prochloraz were 0.0083 µg/ml and 0.002 µg/ml, respectively. No cross-resistance was observed between fungicides from different groups. Mycelial growth rate, control efficacy, and osmotic stress responses were significantly different (P < 0.05) between fludioxonil-sensitive (FluS) and -resistant (FluR) isolates, but no significant difference was observed (P > 0.05) in virulence and sporulation between FluS and FluR isolates. No mutation was detected in coding regions of the CnOs-1, Cal, Hk1, Hog1, TPI, and Mrr1 genes. Interestingly, with fludioxonil treatment, the expression of ABC transporter gene atrB was significantly overexpressed in some resistant isolates. However, overexpression of the atrB gene was not detected in one moderately and one highly resistant isolate, indicating that other unknown mechanisms may be involved. Current findings uncovered several effective chemicals and provided the foundation for designing management strategies to practically control peach anthracnose with the most effective demethylation inhibitor fungicides and quinone outside inhibitor fungicides.

Identification, Genetic Diversity, and Chemical Control of Xanthomonas arboricola pv. pruni in China.

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni has become widespread in most peach-producing areas of China and has caused devastating losses to the peach industry. However, little is known about the population biology and epidemiology of X. arboricola pv. pruni in China, thus no effective management strategy is available. Altogether, 321 symptomatic samples of peach bacterial spot from 12 provinces in China were collected from which 612 bacterial isolates were obtained. Based on 16S rDNA sequence comparison in GenBank, the obtained isolates were identified as Pantoea spp. (514) and Xanthomonas spp. (98). The pathogenicity test demonstrated that the causal agent of the peach bacterial spot was the Xanthomonas spp. instead of the Pantoea spp. Based on morphological observation, physiological and biochemical characterization, and molecular identification, the Xanthomonas spp. were further identified to be X. arboricola pv. pruni. Then, 41 X. arboricola pv. pruni isolates representing different populations were selected and analyzed with repetitive element sequence based-PCR and intersimple sequence repeat markers to understand the genetic diversity and population structure along with four X. arboricola pv. pruni isolates from plum and three isolates of X. arboricola pv. juglandis as comparison. A total of 98 polymorphic alleles were identified, with a mean value of percentage of polymorphic loci of 14. Genetic diversity and phylogenetic analysis revealed the profound heterogeneity between X. arboricola pv. juglandis and X. arboricola pv. pruni, moderate genetic differentiation within X. arboricola pv. pruni, and obvious host specificity but weak geographical differentiation in X. arboricola population. Finally, the efficiency of bactericides on X. arboricola pv. pruni was evaluated in vitro and in vivo. The parallel repeated field trials in two orchards demonstrated that 80% Mancozeb (1:800) and 47% Kocide (1:800, 1:1,500, and 1:2,000) had excellent control efficacies for X. arboricola pv. pruni, especially as the control efficacy of Kocide could even reach 90%. This study conducted a systematic investigation for the occurrence, population variance, and chemical control of X. arboricola pv. pruni. It improved the understanding of the pathogen populations of peach bacterial spot in China and provided solid theoretical and practical guidance for X. arboricola pv. pruni control.

Whole-Genome Sequence of Diaporthe citri Isolate NFHF-8-4, the Causal Agent of Citrus Melanose.

Diaporthe species are the causal agents of melanose, stem-end rot, and gummosis diseases of citrus. D. citri is the predominant species on different citrus varieties. These diseases exceedingly reduce quality and marketability of fresh fruits. Melanose on fruits especially causes massive economic losses. The infection mechanisms of D. citri are still unclear and the genome sequence of D. citri has not been released. In order to systemically explore the interaction between citrus and D. citri, we sequenced the whole-genome of D. citri NFHF-8-4, which was isolated from a sample with melanose in Jiangxi Province. The NFHF-8-4 genome sequence will provide valuable information for studying the development process, infection process, and resistance to fungicides mechanisms in D. citri.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Sequence of Venturia carpophila, the Causal Agent of Peach Scab.

Venturia carpophila, the causal agent of scab disease on peach, is a host-specific fungus that is widely distributed around the world, including China. In our previous study, samples were collected from 14 provinces in China, and 750 isolates were obtained by single-spore separation. Here, we reported the first highly contiguous whole-genome sequence (35.87 Mb) of the V. carpophila isolate ZJHZ1-1-1, which included 33 contigs with N50 value of 2.01 Mb and maximum contig length of 3.39 Mb. The high-quality genome sequence and annotation resource will be useful to study the fungal biology, pathogen-host interaction, fungicide resistance, characterization of important genes, population genetic diversity, and development of molecular markers for genotyping and species identification.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effect of Chemical Seed Treatment on Rice False Smut Control in Field.

Rice false smut, caused by the pathogen Ustilaginoidea virens, is a severe emerging disease in China. It affects not only the quality of rice but also yields of rice production. To make clear the effect of chemical seed treatment on the rice false smut control in fields, during 2014 to 2017, four fungicides with different modes of action were used to treat rice seeds contaminated by false smut balls. In rice-growing seasons, samples of rice tissues were taken for detection of U. virens by using a specific nested PCR method at different rice-growing stages. In addition, the occurrence of rice false smut was investigated at maturation stage. Results showed that U. virens in plant tissues decreased significantly at the seedling stage upon chemical seed treatment. Four chemical treatments decreased the detection rate significantly (P < 0.01) compared with the water treatment, but no significant difference was observed among four chemical treatments. However, the detection rate did not decease significantly at the tillering and booting stages. Similarly, the final occurrence of rice false smut did not show significant difference between each chemical and water treatment. These results suggested that chemical seed treatment had only limited efficacy in preventing occurrence of rice false smut; application of fungicides at the booting stage or integrated use of fungicides and agricultural practices might give a better control for this disease.

Sensitivity of Colletotrichum fructicola and Colletotrichum siamense of Peach in China to Multiple Classes of Fungicides and Characterization of Pyraclostrobin-Resistant Isolates.

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including Colletotrichum fructicola and Colletotrichum siamense, is a devastating disease of peach. Chemical control has been widely used for years, but management failures have increased with the commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, the sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz, and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance, 26% showed low resistance, and all of the C. siamense isolates showed low resistance. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin even when they are members of the same quinone outside inhibitor (QoI) fungicide group or between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for the mycelial growth rates that were detected between high- and low-resistance isolates of C. fructicola. Molecular characterization of the Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the resistance status of C. fructicola and C. siamense to different fungicides and briefly discussed implications of that resistance. Demethylation inhibitor fungicides were found to be the best option among the different chemicals studied here, to control peach anthracnose in China.

Sensitivity of Venturia carpophila from China to Five Fungicides and Characterization of Carbendazim-Resistant Isolates.

Peach scab is a fungal disease caused by Venturia carpophila that can significantly reduce peach yield and quality. Fungicide application is the main control measure for peach scab worldwide. To better understand the fungicide-resistance status and devise suitable management strategies, the sensitivity of 135 single-spore V. carpophila isolates to the commonly used fungicides carbendazim, iprodione, propiconazole, azoxystrobin, and boscalid were determined using a microtiter plate test method. Results showed that the mean effective concentrations to cause inhibitions by 50% (EC50) of tested isolates to iprodione, propiconazole, azoxystrobin, and boscalid were 16.287, 0.165, 0.570, and 0.136 µg/ml, respectively. The EC50 values of V. carpophila isolates to four fungicides displayed unimodal frequency distributions, indicating no resistance occurred to these fungicides. On the contrary, bimodal frequency distribution was observed for carbendazim, indicating that V. carpophila developed resistance to carbendazim. Resistance was widely detected from all 14 provinces studied. Molecular analysis showed that the point mutation E198K of the TUB2 gene determined high resistance, whereas E198G conferred moderate resistance. Moderate and high resistances were stable, and the resistant isolates did not show significant fitness penalties. On the contrary, some resistant isolates showed better competitiveness under certain stresses. This is the first report to detect the sensitivity of V. carpophila to fungicides, which enables future monitoring of fungicide resistance and provides basic information to allow the design of suitable peach scab management strategies.

Rice false smut fungus hijacks the rice nutrients supply by blocking and mimicking the fertilization of rice ovary.

Rice false smut disease is an increasing threat to rice production in the world. Despite of best efforts, research for the infection of the fungus has yielded equivocal and conflicting results about where and how the infection is initiated and developed. Here we show a stepwise infection pattern and sophisticated regulation during this process. Initial infection occurred on the filaments, which prevented the production of mature pollen thus blocked the pollination. In the following days, the pathogen invaded the stigmas and styles, occasionally the ovaries. Expression analysis indicated that the fungus mimicked a successful fertilization process and enabled the continuous supply of nutrients for fungus to produce false smut balls. The stepwise infection of flower organs and mimicry of ovary fertilization unveiled in this study guided the rice plant into supplying nutrients for false smut ball development and represents a new and unique biological process of host pathogen interactions.

Fitness and Competitive Ability of Alternaria alternata Field Isolates with Resistance to SDHI, QoI, and MBC Fungicides.

Field isolates of Alternaria alternata from peach were previously characterized for their sensitivity to succinate dehydrogenase inhibitor (SDHI) fungicides and the underlying molecular basis of resistance was determined. In the present study, we report that isolates resistant to the SDHI fungicide boscalid, regardless of genotype, were also resistant to pyraclostrobin and thiophanate-methyl. Resistance to pyraclostrobin was due to the G143A mutation in cytochrome b and resistance to thiophanate-methyl was due to 167Y in ß-tubulin. Representatives of the two most commonly isolated SDHI resistance genotypes, H277Y in sdh subunit B and H134R in sdh subunit C, as well as genotype D123E in sdh subunit D, were selected for fitness evaluations. Genotypes H277Y and H134R suffered no fitness penalties based on mycelial growth on potato dextrose agar, spore production in vitro, osmotic sensitivity, oxidative sensitivity, germination ability, or the ability to cause disease on peach fruit. Hypersensitivity to oxidative stress and weak sporulation was observed only in genotype D123E. No competitive advantage was detected for sensitive isolates over the course of five consecutive transfers on peach fruit when spores were mixed with genotypes H277Y or H134R. Results suggest that, in the absence of fungicide pressure, A. alternata isolates resistant to methyl benzimidazole carbamate, quinone outside inhibitor, and SDHI fungicides carrying the H277Y mutation in SDHB and the H134R mutation in SDHC may effectively compete with the boscalid-sensitive populations.

Identification and Characterization of Three Monilinia Species from Plum in China.

In total, 112 Monilinia spp. single-spore isolates were collected from plum fruit (Prunus salicina) symptomatic for brown rot disease from Yunnan, Hubei, and Zhejiang provinces and Chongqing municipality, China between 2012 and 2014. Three distinct colony morphologies (phenotypes) were observed on potato dextrose agar and two isolates per phenotype were selected for further analysis. Colony color, colony shape, conidia size, number of germ tubes per conidia, and pathogenicity on plum were investigated. The ribosomal internal transcribed spacer regions 1 and 2 as well as a polymerase chain reaction-based method that amplified fragments of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and ß-tubulin (TUB2) genes were used to identify the isolates to the species level. The three phenotypes were identified to be three different species: Monilinia fructicola, Monilia mumecola, and Monilia yunnanensis. Phylogenetic analysis based on G3PDH and TUB2 nucleotide sequences revealed that isolates within species clustered together regardless of host or geographical origin, suggesting that these factors did not play an important role for the evolutionary separation of the described species.