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 soil-borne, 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 down-regulated, whereas many genes involved in metal iron binding were up-regulated in the Δfpkapc strain, compared with the wild type. 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 wild type. Also, relative to WT, the Δfpkapc strain showed greater replaced by increased tolerance to ion stress, but decreased tolerance to H2O2. The mutant caused smaller disease lesions on wheat and barley plants, but the significantly increased TRI genes expression, compared with the wild type. In summary, Fpkapc plays multiple roles in governing growth, development, stress responses, and virulence in F. pseudograminearum.

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 dsRNA 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 that 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-. And 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 hours 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.

Trichoderma longibrachiatum (TG1) Enhances Wheat Seedlings Tolerance to Salt Stress and Resistance to Fusarium pseudograminearum

Salinity is abiotic stress that inhibits seed germination and suppresses plant growth and root development in a dose-dependent manner. Fusarium pseudograminearum (Fg) is a plant pathogen that causes wheat crown rot. Chemical control methods against Fg are toxic to the environment and resistance has been observed in wheat crops. Therefore, an alternative approach is needed to manage this devastating disease and the effects of salinity. Our research focused on the mycoparasitic mechanisms of Trichoderma longibrachiatum (TG1) on Fg and the induction of defenses in wheat seedlings under salt and Fg stress at physiological, biochemical and molecular levels. The average inhibition rate of TG1 against Fg was 33.86%, 36.32%, 44.59%, and 46.62%, respectively, in the four NaCl treatments (0, 50, 100, and 150 mM). The mycoparasitic mechanisms of TG1 against Fg were coiling, penetration, and wrapping of Fg hyphae. In response to inoculation of TG1 with Fg, significant upregulation of cell wall degrading enzymes (CWDEs) was observed. The expression of β-1, 6-glucan synthase (PP4), endochitinase precursor (PH-1), and chitinase (chi18-15) increased by 1. 6, 1. 9, and 1.3-fold on day 14 compared with day 3. Wheat seedlings with combined TG1 + Fg treatments under different NaCl stress levels decreased disease index by an average of 51.89%; increased the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity by an average of 38%, 61%, and 24.96%, respectively; and decreased malondialdehyde (MDA) and hydrogen peroxide (H2O2) content by an average of 44.07% and 41.75% respectively, compared with Fg treated seedlings. The combined TG1 + Fg treatment induced the transcription level of plant defense-related genes resulting in an increase in tyrosin-protein kinase (PR2), chitinase class I (CHIA1), and pathogenesis-related protein (PR1-2) by an average of 1.15, 1.35, and 1.37-fold, respectively compared to Fg treatment. However, the expression levels of phenylalanine ammonia-lyase (PAL) increased 3.40-fold under various NaCl stresses. Our results suggest that TG1 enhances wheat seedling growth and controls wheat crown rot disease by strengthening the plant defense system and upregulating the expression of pathogenesis-related genes under both Fg and salt stress.

The Wheat Wall-Associated Receptor-Like Kinase TaWAK-6D Mediates Broad Resistance to Two Fungal Pathogens Fusarium pseudograminearum and Rhizoctonia cerealis

The soil-borne fungi Fusarium pseudograminearum and Rhizoctonia cerealis are the major pathogens for the economically important diseases Fusarium crown rot (FCR) and sharp eyespot of common wheat (Triticum aestivum), respectively. However, there has been no report on the broad resistance of wheat genes against both F. pseudograminearum and R. cerealis. In the current study, we identified TaWAK-6D, a wall-associated kinase (WAK) which is an encoding gene located on chromosome 6D, and demonstrated its broad resistance role in the wheat responses to both F. pseudograminearum and R. cerealis infection. TaWAK-6D transcript induction by F. pseudograminearum and R. cerealis was related to the resistance degree of wheat and the gene expression was significantly induced by exogenous pectin treatment. Silencing of TaWAK-6D compromised wheat resistance to F. pseudograminearum and R. cerealis, and repressed the expression of a serial of wheat defense-related genes. Ectopic expression of TaWAK-6D in Nicotiana benthamiana positively modulated the expression of several defense-related genes. TaWAK-6D protein was determined to localize to the plasma membrane in wheat and N. benthamiana. Collectively, the TaWAK-6D at the plasma membrane mediated the broad resistance responses to both F. pseudograminearum and R. cerealis in wheat at the seedling stage. This study, therefore, concludes that TaWAK-6D is a promising gene for improving wheat broad resistance to FCR and sharp eyespot.

Heat Stress Tolerance Gene FpHsp104 Affects Conidiation and Pathogenicity of Fusarium pseudograminearum

Heat shock protein Hsp104, a homolog of the bacterial chaperone ClpB and plant Hsp100, plays an essential part in the response to heat and various chemical agents in Saccharomyces cerevisiae. However, their functions remain largely unknown in plant fungal pathogens. Here, we report the identification and functional characterization of a plausible ortholog of yeast Hsp104 in Fusarium pseudograminearum, which we termed FpHsp104. Deletion mutant of FpHsp104 displayed severe defects in the resistance of heat shock during F. pseudograminearum mycelia and conidia when exposed to extreme heat. We also found that the protein showed dynamic localization to small particles under high temperature. However, no significant differences were detected in osmotic, oxidative, or cell wall stress responses between the wild-type and Δfphsp104 strains. Quantitative real-time PCR analysis showed that FpHsp104 was upregulated in the conidia, and disruption of FpHsp104 gene resulted in defects in conidia production, morphology, and germination. The transcript levels of conidiation-related genes of FpFluG, FpVosA, FpWetA, and FpAbaA were reduced in the Δfphsp104 mutant vs. the wild-type strain, but heat-shocked mRNA splicing repair was not affected in Δfphsp104. Moreover, Δfphsp104 mutant also showed attenuated virulence, but its DON synthesis was normal. These data from the first study of Hsp104 in F. pseudograminearum strongly suggest that FpHsp104 gene is an important element in the heat tolerance, development, and pathogenicity processes of F. pseudograminearum.

Developing Actinobacterial Endophytes as Biocontrol Products for Fusarium pseudograminearum in Wheat

Crown rot of wheat, caused by Fusarium pseudograminearum, results in millions of dollars of yield losses globally each year. Management strategies to control crown rot are limited and there are concerns about development of fungicide resistance so novel treatment strategies are desirable. A collection of endophytic Actinobacteria was screened for their ability to suppress the growth of F. pseudograminearum and the development of crown rot symptoms in wheat with the aim of identifying candidates that can be developed into biocontrol products. The ability of the Actinobacteria isolates to suppress the growth of three different F. pseudograminearum strains in vitro was assessed using agar-plate competition assays. Soil-free seedling assays were used to screen for suppression of development of early disease symptoms in the susceptible wheat (Triticum aestivum) cv. Tamaroi. Four of the isolates were tested in a glasshouse pot experiment to assess their ability to decrease disease symptoms and prevent yield losses in wheat cv. Tamaroi grown to maturity in an unsterilized soil. The screening of 53 isolates identified two Streptomyces isolates, MH71 and MH243, with very strong antifungal activity against F. pseudograminearum strains in agar-plate competition and seedling assays. In the glasshouse pot trial, plants treated with seed coatings of either MH71 or MH243 had > 24% lower disease severity than control plants infected with F. pseudograminearum. These two cultures show potential for development as biocontrol products because they are easy to culture, grow on relatively inexpensive media, produce highly durable spores and can be delivered to plants as a seed coat.

Investigating the Impact of Root-Lesion Nematodes (Pratylenchus thornei) and Crown Rot (Fusarium pseudograminearum) on Diverse Cereal Cultivars in a Conservation Farming System

In two experiments on a farm practicing conservation agriculture, the grain yield of a range of wheat cultivars was significantly (p < 0.001) negatively related to the post-harvest population densities of Pratylenchus thornei in the soil profile to 45 cm depth. In a third and fourth experiment with different rotations, methyl bromide fumigation significantly (p < 0.05) decreased (a) a low initial population density of P. thornei in the soil profile to 90 cm depth and (b) a high initial population of P. thornei to 45 cm depth, and a medium level of the crown rot fungus, Fusarium pseudograminearum, at 0–15 cm depth to a low level. For a range of wheat and durum cultivars, grain yield and response to fumigation were highly significantly (p < 0.001) related to (a) the P. thornei tolerance index of the cultivars in the third experiment, and (b) to both the P. thornei tolerance index and the crown rot resistance index in the fourth experiment. In the latter, grain yield was significantly (p < 0.001) positively related to biomass at anthesis and negatively related to percentage whiteheads at grain fill growth stage. One barley cultivar was more tolerant to both diseases than the wheat and durum cultivars. Crop rotation, utilizing crop cultivars resistant and tolerant to both P. thornei and F. pseudograminearum, is key to success for conservation farming in this region.