Survey of prothioconazole sensitivity in Fusarium pseudograminearum isolates from Henan Province, China, and characterization of resistant laboratory mutants.

BACKGROUND: Fusarium crown rot (FCR) is one of the most significant diseases limiting crop production in the Huanghuai wheat-growing region of China. Prothioconazole, a triazole sterol 14α-demethylation inhibitor (DMI) fungicide developed by the Bayer Crop Protection Company, is mainly registered for the prevention and control of wheat powdery mildew and stripe rust (China Pesticide Information Network). It is known to exhibit high activity against F. pseudograminearum, but further research, particularly regarding the potential for fungicide resistance, is required before it can be registered for the control of FCR in China. RESULTS: The current study found that the baseline sensitivity of 67 field isolates of F. pseudograminearum collected between 2019 and 2021 ranged between 0.016-2.974 µg/mL, with an average EC50 value of 1.191 ± 0.720 µg/mL (mean ± SD). Although none of the field isolates exhibited signs of resistance, three highly resistant mutants were produced by repeated exposure to prothioconazole under laboratory conditions. All of the mutants were found to exhibit significantly reduced growth rates on potato dextrose agar (PDA), as well as reduced levels of sporulation, which indicated that there was a fitness cost associated with the resistance. However, inoculation of wounded wheat coleoptiles revealed that the pathogenicity of the resistant mutants was little affected or actually increased. Molecular analysis of the genes corresponding to the prothioconazole target protein, FpCYP51 (FpCYP51A, FpCYP51B, and FpCYP51C), indicated that the resistant mutants contained three conserved substitutions (M63I, A205S, and I246V) that were present in the FpCYP51C sequence of all three mutants, as well as several non-conserved substations in their FpCYP51A and FpCYP51B sequences. Expression analysis revealed that the presence of prothioconazole (0.1 µg/mL) generally resulted in reduced expression of the three FpCYP51 genes, but that the three mutants exhibited more complex patterns of expression that differed in comparison to their parental isolates. The study found no evidence of cross-resistance between prothioconazole and any of the fungicides tested including three DMI fungicides tebuconazole, prochloraz, and flutriafol. CONCLUSIONS: Taken together these results not only provide new insight into the resistant mechanism and biological characteristics associated with prothioconazole resistance in F. pseudograminearum, but also strong evidence that prothioconazole could provide effective and sustained control of FCR, especially when applied in combination with other fungicides.

Analysis of resistance risk and mechanism of the 14α-demethylation inhibitor ipconazole in Fusarium pseudograminearum.

Ipconazole is a broad-spectrum triazole fungicide that is highly effective against Fusarium pseudograminearum. However, its risk of developing resistance and mechanism are not well understood in F. pseudograminearum. Here, the sensitivities of 101 F. pseudograminearum isolates to ipconazole were investigated, and the average EC50 value was 0.1072 µg/mL. Seven mutants resistant to ipconazole were obtained by fungicide adaption, with all but one showing reduced fitness relative to the parental isolates. Cross-resistance was found between ipconazole and mefentrifluconazole and tebuconazole, but none between ipconazole and pydiflumetofen, carbendazim, fludioxonil, or phenamacril. In summary, these findings suggest that there is a low risk of F. pseudograminearum developing resistance to ipconazole. Additionally, a point mutation, G464S, was seen in FpCYP51B and overexpression of FpCYP51A, FpCYP51B and FpCYP51C was observed in ipconazole-resistant mutants. Assays, including transformation and molecular docking, indicated that G464S conferred ipconazole resistance in F. pseudograminearum.

Discovery of Fusarium pseudograminearum Causing Crown Rot of Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Fusarium pseudograminearum is an important plant pathogen that invades many crops (Zhang et al. 2018). Since it was first discovered in Australia in 1951, F. pseudograminearum has been reported in many countries and regions and caused huge economic losses (Burgess et al. 2001). In 2012, crown rot of wheat caused by F. pseudograminearum was discovered for the first time in Henan Province, China (Li et al. 2012). Wheat (Triticum aestivum L.) is one of the most important food crops in Xinjiang Uygur Autonomous Region (XUAR), with 1.07 million hectares cultivated in 2020. In June 2023, a survey of crown rot disease was carried out in winter wheat cv. Xindong 20 in Hotan area, XUAR, China (80.148907°E, 37.051474°N). About 5% of wheat plants showed symptoms of crown rot such as browning of the stem base and white head. The disease was observed in 85% of wheat fields. In order to identify the pathogens, 36 pieces of diseased stem basal tissue, 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, then rinsed three times with sterile water and placed on potato dextrose agar (PDA) medium at 25°C. A total of 27 isolates with consistent morphological characteristics were obtained using single-spore technique (Leslie and Summerell. 2006), and the isolation rate was 75%. The isolates grew rapidly on PDA, produced large numbers of fluffy white hyphae, and pink pigment accumulated in the medium. The isolates were grown on 2% mung bean flour medium and identified by morphological and molecular methods. Macroconidia were abundant, relatively slender, curved to almost straight, commonly two to seven septate, and averaged 22 to 72 × 1.8 to 4.9 µm. Microconidia were not observed. The morphological characters are consistent with Fusarium (Aoki and O'Donnell. 1999). Two isolates (LP-1 and LP-3) were selected for molecular identification. Primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') were used to amplify a portion of the EF-1α gene (O'Donnell et al. 1998). The two 696 bp PCR products were sequenced and submitted to GenBank. The EF-1α gene sequences (GenBank Accession No: PP062794 and PP062795) shared 99.9% identity (695/696) with published F.pseudograminearum sequences (e.g., OP105187, OP105184, OP105179, OP105173). The identification was further confirmed by F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell. 1999). The expected PCR products of 518 bp were produced only in F. pseudograminearum. Pathogenicity tests of LP-1 and LP-3 isolates were performed on 7-day-old seedlings of winter wheat cv. Xindong 20 using the drip inoculation method with a 10-µl of a 106 macroconidia ml-1 suspension near the stem base (Xu et al. 2017). The experiment was repeated five times in a 20 to 25°C greenhouse. Control seedlings were treated with sterile water. After 4 weeks, wheat seedling death and crown browning occurred in the inoculated plants with over 90% incidence. No symptoms were observed in the control plants. The pathogen was reisolated from the inoculated plants by the method described above and identified by morphological and PCR amplification using F. pseudograminearum species-specific primers Fp1-1/Fp1-2. No F. pseudograminearum was isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of winter wheat in XUAR of China. Since F. pseudograminearum can cause great damage to wheat, one of the most important food crops in China, necessary measures should be taken to prevent the spread of F. pseudograminearum to other regions.

Antifungal activities of metconazole against the emerging wheat pathogen Fusarium pseudograminearum.

Fusarium crown rot of wheat is a serious fungal disease that occurs worldwide. The disease has been emerging in the major wheat-growing areas in China since 2010. Fusarium pseudogramineaum is the predominant causative pathogen of crown rot of wheat in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against Fusarium spp., but little is known about its specific activity against F. pseudogramineaum. Metconazole exhibited strong antifungal activities against all thirty-nine F. pseudogramineaum strains collected from the major wheat-growing areas in China. Metconazole inhibited mycelial growth and conidial germ tube elongation of F. pseudograminearum. Metconazole treatment significantly reduced the production of major toxins and the expression levels of toxin biosynthesis genes. Genome-wide transcriptional profiling of F. pseudograminearum in response to metconazole indicated that the expression of genes involved in ergosterol biosynthesis, including fungicide target genes (cyp51 genes), was significantly induced by metconazole. Nine ATP-binding cassette (ABC) transporter-encoding genes were significantly expressed in response to metconazole treatment. Reduced ergosterol production and antioxidant enzyme activities were observed after metconazole treatment. Greenhouse experiments indicated a significant reduction in crown rot occurrence in wheat after seed treatment with metconazole. This study evaluated the potential of metconazole to manage wheat crown rot and provides information to understand its antifungal activities and mechanism of action against F. pseudograminearum.

Resistance risk assessment of Fusarium pseudograminearum from wheat to prothioconazole.

Fusarium crown rot (FCR), primarily caused by Fusarium pseudograminearum, poses significant threats to cereal crops worldwide. Prothioconazole is a demethylation inhibitor (DMI) fungicide used to control FCR. However, the risk of resistance in F. pseudograminearum to prothioconazole has not yet been evaluated. In this study, the sensitivity of a total of 255 F. pseudograminearum strains obtained from Henan Province, China to prothioconazole were determined by the mycelial growth inhibition. The results showed that the effective concentration to 50% growth inhibition (EC50) of these strains ranged from 0.4228 µg/mL to 2.5284 µg/mL, with a mean EC50 value of 1.0692 ± 0.4527 µg/mL (mean ± SD). Thirty prothioconazole-resistant mutants were obtained out of six selected sensitive parental strains by means of fungicide taming. The resistant mutants exhibited defects in vegetative growth, conidia production, and pathogenicity on wheat seedlings compared to their parental strains. Under ion, cell wall, and temperature stress conditions but not osmotic stress, all the mutants exhibited decreased growth rates compared with their parental strains, which was consistent with the control treatment. Cross-resistance test showed that there was a cross-resistance relationship between prothioconazole and four DMI fungicides, including prochloraz, metconazole, tebuconazole and hexaconazole, but no cross-resistance was observed between prothioconazole and carbendazim, phenamacril, fludioxonil, or azoxystrobin. Although no site mutation occurred on Cyp51a and Cyp51b genes, the constitutive expression level of the Cyp51a gene was significantly increased in all mutants. After being treated with prothioconazole, the Cyp51a and Cyp51b genes were significantly increased in both the resistant mutants and their parents. These results suggested that the resistance to prothioconazole of the mutants may be attributed to the changes of the relative expression level of Cyp51a and Cyp51b genes. Taken together, these results could provide a theoretical basis for the scientific use of prothioconazole in the field and fungicide resistance management strategies.

Impact of Phenamacril on the Growth and Development of Fusarium pseudograminearum and Control of Crown Rot of Wheat.

Fusarium pseudograminearum is the dominant pathogen causing Fusarium crown rot (FCR) of wheat. Phenamacril is a 2-cyanoacrylate fungicide, having a control effect on diseases caused by Fusarium spp. The objective of this study was to investigate the inhibitory effect of phenamacril on F. pseudograminearum and its control efficacy against FCR. The results showed that phenamacril had a strong inhibitory effect on the mycelial growth of F. pseudograminearum, EC50 values of phenamacril to 63 tested strains were in the range of 0.0998 to 0.5672 µg/ml, and the average EC50 value was 0.3403 ± 0.0872 µg/ml and could be used as the baseline sensitivity of F. pseudograminearum to phenamacril. Phenamacril reduced the germination rate of conidia of F. pseudograminearum, and the EC50 value was 5.0273 to 26.4814 µg/ml. In addition, we found that phenamacril had a teratogenic effect on conidia and blastotubules, which increased the ratio of conidial germination from the middle cells and showed high efficacy on the sporulation quantity of F. pseudograminearum with an EC50 value in the range of 0.0770 to 0.1064 µg/ml. There was no significant correlation between the sensitivity of F. pseudograminearum to phenamacril and its sensitivity to fludioxonil, carbendazim, tebuconazole, and kresoxim-methyl. In vitro and greenhouse assays showed that the treatment with 0.125 µl of active ingredient per gram recorded the best control effect on wheat crown rot, reaching 87.8 and 77.3%, respectively. In two experimental sites in Luoyang, phenamacril also had great control effect against FCR, reaching 83.9%. It was proven that phenamacril has a superior control effect against FCR. This study has laid a foundation for the study of the mechanism of action of phenamacril against F. pseudograminearum and provided a theoretical basis for the application of phenamacril to control FCR.

Baseline Pydiflumetofen Sensitivity of Fusarium pseudograminearum Isolates Collected from Henan, China, and Potential Resistance Mechanisms.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is one of the most important diseases impacting wheat production in the Huanghuai region, the most important wheat-growing region of China. The current study found that the SDHI fungicide pydiflumetofen, which was recently developed by Syngenta Crop Protection, provided effective control of 67 wild-type F. pseudograminearum isolates in potato dextrose agar, with an average EC50 value of 0.060 ± 0.0098 µg/ml (SE). Further investigation revealed that the risk of fungicide resistance in pydiflumetofen was medium to high. Four F. pseudograminearum mutants generated by repeated exposure to pydiflumetofen under laboratory conditions indicated that pydiflumetofen resistance was associated with fitness penalties. Mutants exhibited significantly (P < 0.05) reduced sporulation in mung bean broth and significantly (P < 0.05) reduced pathogenicity in wheat seedlings. Sequence analysis indicated that the observed pydiflumetofen resistance of the mutants was likely associated with amino acid changes in the different subunits of the succinate dehydrogenase target protein, including R18L and V160M substitutions in the FpSdhA sequence; D69V, D147G, and C257R in FpSdhB; and W78R in FpSdhC. This study found no evidence of cross-resistance between pydiflumetofen and the alternative fungicides tebuconazole, fludioxonil, carbendazim, or fluazinam, which all have distinct modes of action and could therefore be used in combination or rotation with pydiflumetofen to reduce the risk of resistance emerging in the field. Taken together, these results indicate that pydiflumetofen has potential as a novel fungicide for the control of FCR caused by F. pseudograminearum and could therefore be of great significance in ensuring high and stable wheat yields in China.

High-Quality Genome Resource of Fusarium pseudograminearum Isolate Fp22-2 by Oxford Nanopore Long-Read Sequencing.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, results in severe yield and quality losses of cereal crops in many arid and semiarid areas of the world. Limited information about the genome of F. pseudograminearum restricts the pathogenesis research and breeding of disease-resistant wheat varieties. In this study, a high-quality genome assembly of F. pseudograminearum isolate Fp22-2 was generated using Oxford Nanopore long-read sequencing technology. The assembled nuclear genome of Fp22-2 is 37.33 Mb with a repeat content of 3.69% and is divided into four contigs with a k-mer completeness score of 97.2% and a base quality accuracy of >99.99%. A total of 14,475 protein-coding genes (BUSCO completeness score, 99.9%) were predicted and functionally annotated. Moreover, genes encoding pathogenic proteins, including effector proteins and carbohydrate-active enzymes, and secondary metabolic gene clusters were identified. Overall, the high-quality genome assembly and gene annotation provided here will allow further investigation of the biology of F. pseudograminearum and lead to the development of new control options for FCR.

Host Specificity of Soilborne Pathogens in Hordeum Species and Their Relatives.

Soilborne pathogens destabilize the yields of Triticeae crops, including barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). Although genetic resistance derived from relatives of these species has been utilized to prevent rust diseases (i.e., in the wheat-rye 1BL-1RS translocation line), research on resistance against soilborne pathogens remains limited. Here, we performed field trials using 76 genotypes representing 28 Hordeum, six Triticum, and two Aegilops species to examine resistance against three soilborne bymoviruses: barley yellow mosaic virus (BaYMV), barley mild mosaic virus (BaMMV), and wheat yellow mosaic virus (WYMV). We also performed greenhouse tests using the soilborne fungal pathogen Fusarium pseudograminearum, which causes Fusarium crown rot (FCR). Using RT-PCR, we detected BaMMV and BaYMV in several Hordeum species, whereas WYMV induced systemic infection in the Triticum and Aegilops species. The identification of FCR susceptibility in all species examined suggests that F. pseudograminearum is a facultative fungal pathogen in Triticeae. Intraspecies variation in FCR disease severity was observed for several species, pointing to the possibility of exploring host resistance mechanisms. Therefore, by unlocking the host specificity of four soilborne pathogens in Hordeum species and their relatives, we obtained insights for the further exploration of wild sources of soilborne pathogen resistance for future wheat and barley improvement programs.

A Putative Zn(II)2Cys6-Type Transcription Factor FpUme18 Is Required for Development, Conidiation, Cell Wall Integrity, Endocytosis and Full Virulence in Fusarium pseudograminearum.

Fusarium pseudograminearum is one of the major fungal pathogens that cause Fusarium crown rot (FCR) worldwide and can lead to a substantially reduced grain yield and quality. Transcription factors play an important role in regulating growth and pathogenicity in plant pathogens. In this study, we identified a putative Zn(II)2Cys6 fungal-type domain-containing transcription factor and named it FpUme18. The expression of FpUME18 was induced during the infection of wheat by F. pseudograminearum. The ΔFpume18 deletion mutant showed defects in growth, conidial production, and conidial germination. In the responses to the cell wall, salt and oxidative stresses, the ΔFpume18 mutant inhibited the rate of mycelial growth at a higher rate compared with the wild type. The staining of conidia and mycelia with lipophilic dye FM4-64 revealed a delay in endocytosis when FpUME18 was deleted. FpUME18 also positively regulated the expression of phospholipid-related synthesis genes. The deletion of FpUME18 attenuated the pathogenicity of wheat coleoptiles. FpUME18 also participated in the production of the DON toxin by regulating the expression of TRI genes. Collectively, FpUme18 is required for vegetative growth, conidiation, stress response, endocytosis, and full virulence in F. pseudograminearum.

Piriformospora indica Increases Resistance to Fusarium pseudograminearum in Wheat by Inducing Phenylpropanoid Pathway.

Fusarium crown rot (FCR), mainly caused by Fusarium pseudograminearum, not only seriously threatens the yield and quality of wheat, but also endangers the health and safety of humans and livestock. Piriformospora indica is a root endophytic fungus that colonizes plant roots extensively and can effectively promote plant growth and improve plant resistance to biotic and abiotic stresses. In this study, the mechanism of FCR resistance mediated by P. indica in wheat was revealed from the phenylpropanoid metabolic pathway. The results showed that the colonization of P. indica significantly reduced the progression of wheat disease, the amount of F. pseudograminearum colonization, and the content of deoxynivalenol (DON) in wheat roots. RNA-seq suggested that P. indica colonization could reduce the number of differentially expressed genes (DEGs) in the transcriptome caused by F. pseudograminearum infection. The DEGs induced by the colonization of P. indica were partially enriched in phenylpropanoid biosynthesis. Transcriptome sequencing and qPCR indicated that the colonization of P. indica up-regulated the expression of genes involved in the phenylpropanoid biosynthesis pathway. The metabolome analysis indicated that the colonization of P. indica increased the metabolites' accumulation in the phenylpropanoid biosynthesis. Consistent with transcriptome and metabolomic analysis, microscopic observations showed enhanced lignin accumulation in the roots of the Piri and Piri+Fp lines, most likely contributing to the arrested infection by F. pseudograminearum. These results suggested that P. indica increased resistance to F. pseudograminearum in wheat by inducing the phenylpropanoid pathway.

The Cytosolic Acetoacetyl-CoA Thiolase TaAACT1 Is Required for Defense against Fusarium pseudograminearum in Wheat.

Fusarium pseudograminearum is a major pathogen for the destructive disease Fusarium crown rot (FCR) of wheat (Triticum aestivum). The cytosolic Acetoacetyl-CoA thiolase II (AACT) is the first catalytic enzyme in the mevalonate pathway that biosynthesizes isoprenoids in plants. However, there has been no investigation of wheat cytosolic AACT genes in defense against pathogens including Fusarium pseudograminearum. Herein, we identified a cytosolic AACT-encoding gene from wheat, named TaAACT1, and demonstrated its positively regulatory role in the wheat defense response to F. pseudograminearum. One haplotype of TaAACT1 in analyzed wheat genotypes was associated with wheat resistance to FCR. The TaAACT1 transcript level was elevated after F. pseudograminearum infection, and was higher in FCR-resistant wheat genotypes than in susceptible wheat genotypes. Functional analysis indicated that knock down of TaAACT1 impaired resistance against F. pseudograminearum and reduced the expression of downstream defense genes in wheat. TaAACT1 protein was verified to localize in the cytosol of wheat cells. TaAACT1 and its modulated defense genes were rapidly responsive to exogenous jasmonate treatment. Collectively, TaAACT1 contributes to resistance to F. pseudograminearum through upregulating the expression of defense genes in wheat. This study sheds new light on the molecular mechanisms underlying wheat defense against FCR.

A Novel Wall-Associated Kinase TaWAK-5D600 Positively Participates in Defense against Sharp Eyespot and Fusarium Crown Rot in Wheat.

Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. In this study, we performed a genome-wide analysis of wall-associated kinase (WAK) family in wheat. As a result, a total of 140 TaWAK (not TaWAKL) candidate genes were identified from the wheat genome, each of which contains an N-terminal signal peptide, a galacturonan binding domain, an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular Serine/Threonine protein kinase domain. By analyzing the RNA-sequencing data of wheat inoculated with R. cerealis and F. pseudograminearum, we found that transcript abundance of TaWAK-5D600 (TraesCS5D02G268600) on chromosome 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Importantly, knock-down of TaWAK-5D600 transcript impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum, and significantly repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. Thus, this study proposes TaWAK-5D600 as a promising gene for improving wheat broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat.

A Megabirnavirus Alleviates the Pathogenicity of Fusarium pseudograminearum to Wheat.

Fusarium pseudograminearum is a phytopathogen that causes wheat crown rot disease worldwide. Fusarium pseudograminearum megabirnavirus 1 (FpgMBV1) was isolated from the hypovirulent strain FC136-2A of F. pseudograminearum as a novel double-stranded RNA mycovirus belonging to the family Megabirnaviridae. Here we examined the effects of FpgMBV1 on colony morphology and pathogenicity of F. pseudograminearum. Through hyphal tip culture, we obtained virus-free progeny of strain FC136-2A, referred to as FC136-2A-V-. FpgMBV1 was transferred horizontally to another virus-free strain, WZ-8A-HygR-V-. The progeny obtained through horizontal transfer was referred to as WZ-8A-HygR-V+. Colony morphology was similar between the FpgMBV1-positive and -negative strains. The ability to penetrate cellophane in vitro was lost, and pathogenicity on wheat plants was reduced significantly in the FpgMBV1-positive strains relative to the FpgMBV1-negative strains. Microscopic observations showed a 6-h delay in the formation of appressoria-like structures in FC136-2A relative to FC136-2A-V-. Mycelium extension was significantly longer in wheat coleoptiles infected by WZ-8A-HygR-V- than in that infected by WZ-8A-HygR-V+ at 12 and 20 h after inoculation (hai). In addition, expression of five genes that encode cell wall-degrading enzymes differed significantly between FpgMBV1-positive and -negative strains at 12 and 20 hai during early infection of wheat cells by conidia. This study provides evidence for the hypovirulence effect of FpgMBV1 on F. pseudograminearum and suggests that the underlying mechanism involves unsuccessful early infection and perhaps cell wall degradation.

Transcriptome Analysis and Functional Validation Identify a Putative bZIP Transcription Factor, Fpkapc, that Regulates Development, Stress Responses, and Virulence in Fusarium pseudograminearum.

Fusarium pseudograminearum is a soilborne, hemibiotrophic phytopathogenic fungus that causes Fusarium crown rot and Fusarium head blight in wheat. The basic leucine zipper proteins (bZIPs) are evolutionarily conserved transcription factors that play crucial roles in a range of growth and developmental processes and the responses to biotic and abiotic stresses. However, the roles of bZIP transcription factors remains unknown in F. pseudograminearum. In this study, a bZIP transcription factor Fpkapc was identified to localize to the nucleus in F. pseudograminearum. A mutant strain (Δfpkapc) was constructed to determine the role of Fpkapc in growth and pathogenicity of F. pseudograminearum. Transcriptomic analyses revealed that many genes involved in basic metabolism and oxidation-reduction processes were downregulated, whereas many genes involved in metal iron binding were upregulated in the Δfpkapc strain, compared with the wild type (WT). Correspondingly, the mutant had severe growth defects and displayed abnormal hyphal tips. Conidiation in the Fpkapc mutant was reduced, with more conidia in smaller size and fewer septa than in the WT. Also, relative to WT, the Δfpkapc strain showed greater tolerance to ion stress, but decreased tolerance to H2O2. The mutant caused smaller disease lesions on wheat and barley plants, but significantly increased TRI gene expression, compared with the WT. In summary, Fpkapc plays multiple roles in governing growth, development, stress responses, and virulence in F. pseudograminearum.

Identification and Characterization of the Homeobox Gene Family in Fusarium pseudograminearum Reveal Their Roles in Pathogenicity.

Homeobox transcription factors have been implicated in filamentous growth, conidia formation and virulence in fungal pathogens. However, the presence of the homeobox gene family and their potential influence on pathogenesis in Fusarium pseudograminearum have not been investigated. F. pseudograminearum is an important plant pathogen that causes wheat and barley crown rot. In this study, we performed a genome-wide survey for F. pseudograminearum homeobox genes, and 11 FpHtfs were identified and characterized. Domain analyses revealed that all of these proteins contain a complete homeobox domain that contains three helices. Expression profiles of FpHtf genes at different pathogen stages showed that six FpHtf genes were induced during infection. Further, we generated and characterized FpHtf3 deletion mutants in F. pseudograminearum, showing it was essential for virulence. These results indicated that members of the homeobox gene family are likely involved in F. pseudograminearum pathogenicity. Our work also provides a useful foundation for further studies on the complexity and function of the homeobox gene family in F. pseudograminearum.

First Report of Maize Seedling Blight Caused by Fusarium pseudograminearum in China.

Maize (Zea mays L.) is one of the most important food and feed crops in China, with a cultivation area of more than 40 million hectares (http://www.fao.org/faostat/en/#data/QC). In July 2021, a serious maize seeding blight occurred in Changjia Town, Gaoqing Country, Zibo City, Shandong Province, China, and the disease incidence was up to 50% in some fields. The root system of infected plants displayed poor development. The primary roots were brown and rotted. The leaves at the base of the plants were drying up, then the whole plant withered. To determine the cause agent of the disease, symptomatic roots of diseased seedlings were collected and surface-sterilized (70% ethanol for 30 s and 3% sodium hypochlorite (NaClO) for 90 s), subsequently rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), then incubated at 25°C for 2 days. Two cultures with similar morphological characteristics were purified through single-spore isolation technique and identified by morphology and molecular methods as Fusarium pseudograminearum O'Donnell & T. Aoki 1999. Plentiful macroconidia formed in 5-day-old carboxymethyl cellulose (CMC) cultures; microconidia were absent. Macroconidia were thick-walled and curved, usually 3- to 5- septa, 31.6 ± 0.6 µm × 4.8 ± 0.1 µm (n = 50). Colony pigmentation on PDA was pink to red, with white to pink aerial mycelia on PDA cultures was abundant and filled the petri dishes. For molecular identification, the rDNA internal transcribed spacer (ITS) gene and translation elongation factor 1 alpha (TEF-1α) gene of two isolates (SAIA41B and SAIA41C) were amplified with ITS1/ITS4 (White et al., 1990) and EF-1/EF-2 (O'Donnell et al., 1998), respectively. Blastn analysis of both the ITS sequence (accession numbers OM108101 and OM108102) and TEF-1α sequence (accession numbers OM142205 and OM142206) revealed 100% (481/481 bp for ITS and 637/637 bp for TEF-1α) sequence identity with the sequences of F. pseudograminearum reported in GenBank (MW699613 for ITS and JN862232 for TEF-1α). The molecular identification was further confirmed by the F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell 1999). The expected 523-bp fragments were obtained for isolates SAIA41B and SAIA41C. In the pathogenicity test, healthy germinating maize roots (Zhengdan958) were inoculated with PDA culture blocks of isolate SAIA41C. Plants inoculated only with PDA culture blocks served as controls. Maize plants were put in petri dishes and placed in an incubator with a 12-h photoperiod at 25 oC and 100% relative humidity. Seven days later, roots of the plants inoculated with isolate SAIA41C were poorly developed and became brown necrotic and rotted, which were identical to the symptoms observed in the fields, whereas the roots of control plants were developed normally. The pathogen was re-isolated from the necrotic tissue of the inoculated roots but not from the control plants, and its identity was confirmed by PCR with the primes Fp1-1/Fp1-2 described above, fulfilling Koch's Postulates. To our knowledge, this is the first report of maize seedling blight caused by F. pseudograminearum in China. Our finding indicates the potential spread of F. pseudograminearum on maize, and more attention should be paid to prevention and control of maize seedling blight caused by F. pseudograminearum. The author(s) declare no conflict of interest. Acknowledgements: This research was supported by National Natural Science Foundation of China (No. 32102181), Shandong Provincial Natural Science Foundation (No. ZR2021QC059), Wheat Industry Technology System of Shandong Province (No. SDAIT-01-10), and Agricultural Science and Technology Innovation Project of SAAS (No. CXGC2021A38 and CXGC2021A33).

Baseline Sensitivity and Potential Resistance Mechanisms for Fusarium pseudograminearum to Fludioxonil.

Fusarium crown rot (FCR), which is caused by Fusarium pseudograminearum, is one of the most important diseases affecting wheat production in the Huanghuai wheat-growing region of China. Although the phenylpyrrole fungicide fludioxonil is known to have a broad-spectrum activity against a wide range of plant pathogens, including F. pseudograminearum, it has not yet been registered for the control of FCR in China, and further research is needed to assess the biological characteristics and molecular mechanisms associated with fludioxonil resistance, and especially the potential for highly resistant isolates to emerge. The current study demonstrated that the baseline fludioxonil sensitivity of 61 F. pseudograminearum isolates collected from the Henan province of China during the summers of 2019 to 2021 conformed to a unimodal distribution with a mean effective concentration for 50% inhibition (EC50) value of 0.021 ± 0.003 µg/ml, which indicated that none of the isolates exhibited natural resistance to fludioxonil. Nevertheless, four fludioxonil-resistant mutants were attained after repeated exposure to fludioxonil under laboratory conditions. All resistant mutants exhibited significantly lower growth rates on potato dextrose agar (PDA) and lower levels of sporulation and pathogenicity in wheat seedlings. In addition, the resistant mutants also exhibited less growth on PDA amended with either 0.5 M mannitol, 0.5 M glucose, 0.5 M MgCl2, or 0.5 M NaCl, which indicated that they had greater sensitivity to osmotic stress. Molecular analysis of the proposed fludioxonil target protein FpOs1 indicated that the predicted sequences of the resistant mutants contained none of the characteristic amino acid changes previously associated with fludioxonil resistance in other species. Further investigation via quantitative real-time PCR analysis revealed that expression of the FpOs1 gene was significantly altered in the resistant mutants in both the absence and presence of fludioxonil. Meanwhile, plate assays found evidence of cross-resistance between fludioxonil and cyprodinil, as well as with the triazole fungicides tebuconazole and difenoconazole, but not with other commonly used fungicides including prochloraz, fluazinam, and carbendazim. Taken together, these results provide new insights into the mechanism and biological characteristics associated with fludioxonil resistance in F. pseudograminearum and indicate that fludioxonil could provide effective and sustained control of FCR during wheat production.

Peroxisome Proliferator FpPEX11 Is Involved in the Development and Pathogenicity in Fusarium pseudograminearum.

Fusarium crown rot (FCR) of wheat, an important soil-borne disease, presents a worsening trend year by year, posing a significant threat to wheat production. Fusarium pseudograminearum cv. b was reported to be the dominant pathogen of FCR in China. Peroxisomes are single-membrane organelles in eukaryotes that are involved in many important biochemical metabolic processes, including fatty acid ß-oxidation. PEX11 is important proteins in peroxisome proliferation, while less is known in the fungus F. pseudograminearum. The functions of FpPEX11a, FpPEX11b, and FpPEX11c in F. pseudograminearum were studied using reverse genetics, and the results showed that FpPEX11a and FpPEX11b are involved in the regulation of vegetative growth and asexual reproduction. After deleting FpPEX11a and FpPEX11b, cell wall integrity was impaired, cellular metabolism processes including active oxygen metabolism and fatty acid ß-oxidation were significantly blocked, and the production ability of deoxynivalenol (DON) decreased. In addition, the deletion of genes of FpPEX11a and FpPEX11b revealed a strongly decreased expression level of peroxisome-proliferation-associated genes and DON-synthesis-related genes. However, deletion of FpPEX11c did not significantly affect these metabolic processes. Deletion of the three protein-coding genes resulted in reduced pathogenicity of F. pseudograminearum. In summary, FpPEX11a and FpPEX11b play crucial roles in the growth and development, asexual reproduction, pathogenicity, active oxygen accumulation, and fatty acid utilization in F. pseudograminearum.

BdGUCD1 and Cyclic GMP Are Required for Responses of Brachypodium distachyon to Fusarium pseudograminearum in the Mechanism Involving Jasmonate.

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important signaling molecule in plants. cGMP and guanylyl cyclases (GCs), enzymes that catalyze the synthesis of cGMP from GTP, are involved in several physiological processes and responses to environmental factors, including pathogen infections. Using in vitro analysis, we demonstrated that recombinant BdGUCD1 is a protein with high guanylyl cyclase activity and lower adenylyl cyclase activity. In Brachypodium distachyon, infection by Fusarium pseudograminearum leads to changes in BdGUCD1 mRNA levels, as well as differences in endogenous cGMP levels. These observed changes may be related to alarm reactions induced by pathogen infection. As fluctuations in stress phytohormones after infection have been previously described, we performed experiments to determine the relationship between cyclic nucleotides and phytohormones. The results revealed that inhibition of cellular cGMP changes disrupts stress phytohormone content and responses to pathogen. The observations made here allow us to conclude that cGMP is an important element involved in the processes triggered as a result of infection and changes in its levels affect jasmonic acid. Therefore, stimuli-induced transient elevation of cGMP in plants may play beneficial roles in priming an optimized response, likely by triggering the mechanisms of feedback control.