Trichoderma hamatum is a filamentous fungus that serves as a biological control agent for multiple phytopathogens and as an important resource promising for fungicides. However, the lack of adequate knockout technologies has hindered gene function and biocontrol mechanism research of this species. This study obtained a genome assembly of T. hamatum T21, with a 41.4 Mb genome sequence comprising 8170 genes. Based on genomic information, we established a CRISPR/Cas9 system with dual sgRNAs targets and dual screening markers. CRISPR/Cas9 plasmid and donor DNA recombinant plasmid were constructed for disruption of the Thpyr4 and Thpks1 genes. The result indicates the consistency between phenotypic characterization and molecular identification of the knockout strains. The knockout efficiencies of Thpyr4 and Thpks1 were 100% and 89.1%, respectively. Moreover, sequencing revealed fragment deletions between dual sgRNA target sites or GFP gene insertions presented in knockout strains. The situations were caused by different DNA repair mechanisms, nonhomologous end joining (NHEJ), and homologous recombination (HR). Overall, we have successfully constructed an efficient and convenient CRISPR/Cas9 system in T. hamatum for the first time, which has important scientific significance and application value for studies on functional genomics of Trichoderma and other filamentous fungi.

Snow pea root rot in China is caused by Fusarium solani (FSH) and Fusarium avenaceum (FAH), which affect snow pea production. The chemical control methods used against FSH and FAH are toxic to the environment and resistance may be developed in persistence applications. Therefore, an alternative approach is needed to control these pathogens. This study focuses on Trichoderma longibrachiatum strains (TL6 and TL13), mycoparasitic mechanisms of FSH and FAH, as well as growth-promoting potentials on snow pea seedlings under FSH and FAH stress at the physiological, biochemical, and molecular levels. The average inhibitory rates of TL6 against FSH and FAH were 54.58% and 69.16%, respectively, on day 7. Similarly, TL13 average inhibitory rates against FSH and FAH were 59.06% and 71.27%, respectively, on day 7. The combined TL13 and TL6 with FSH and FAH reduced disease severity by 86.6, 81.6, 57.60, and 60.90%, respectively, in comparison to the controls. The snow pea plants inoculated with FSH and FAH without TL6 and TL13 increased malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents in the leaves by 64.8, 66.0, 64.4 and 65.9%, respectively, compared to the control. However, the combined FSH and FAH with TL6 and TL13 decreased the MDA and H₂O₂ content by 75.6, 76.8, 70.0, and 76.4%, respectively, in comparison to the controls. In addition, the combined TL6 + FSH and TL6 + FAH increased the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) by 60.5, 64.7, and 60.3%, respectively, and 60.0, 64.9, and 56.6%, respectively, compared to the controls. Again, compared to the controls, the combined TL13 + FSH and TL13 + FAH increased the activity of SOD, POD, and CAT by 69.7, 68.6, and 65.6%, respectively, and 70.10, 69.5, and 65.8%, respectively. Our results suggest that the pretreatment of snow pea seeds with TL6 and TL13 increases snow pea seedling growth, controls FSH and FAH root rot, increases antioxidant enzyme activity, and activates plant defense mechanisms. The TL13 strain had the greatest performance in terms of pathogen inhibition and snow pea growth promotion compared to the TL6 strain.

Introduction: Root-knot nematode (RKN; Meloidogyne spp.) is one of the most infamous soilborne plant diseases, causing severe crop losses every year. Effector proteins secreted by RKNs play crucial roles during plant-nematode interaction. However, less is known about whether RKN effector proteins can impact the rhizosphere microbial environment.Methods: In this study, we investigated the rhizosphere microbiome community of MiMIF-2 (a plant immunity-modulating effector) transgenic Arabidopsis thaliana with or without nematode infection using the Illumina high-throughput sequencing analysis.Results and discussion: The results showed that the bacterial species richness index increased, while the fungi species richness index decreased in M. incognita-infected MiMIF-2 transgenic A. thaliana plants. The relative abundance of genera such as Clitopilus, Komagataeibacter, Lactobacillus, Prevotella, Moritella, Vibrio, Escherichia-Shigella, and Pseudomonas was reduced in MiMIF-2 transgenic A. thaliana plants compared to wild type, but was significantly increased after inoculation with M. incognita. The Cluster of Orthologous Genes (COG) function classification analysis revealed a decrease in the relative abundance of defense mechanisms, secondary metabolite biosynthesis, transport, and nematode infection catabolism-related functions in MiMIF-2 lines compared to the wild type. These differences may be the reason for the increased susceptibility of MiMIF-2 transgenic A. thaliana to nematode infection. Our results provide a new insight into RKN effector proteins and their association with the microbial community, host, and plant pathogens, which will lead to the exploration of new innovative ideas for future biological control of RKNs.

Meloidogyne enterolobii, a highly virulent and broad-host-range plant-parasitic nematode, poses an increasing threat to global agricultural production. By inducing the formation of nutrient-rich giant cells in host roots and deploying a diverse array of effector proteins to modulate plant immune responses, this nematode achieves efficient colonization and invasion, resulting in impaired crop growth and significant economic losses. In recent years, global climate warming combined with the rapid development of protected agriculture has broken the traditional geographical limits of tropical and subtropical regions, thereby increasing the risk of M. enterolobii occurrence in temperate and high-latitude areas. Concurrently, conventional chemical control methods are increasingly limited by environmental pollution and the development of resistance, steering research toward green control strategies. This review systematically summarizes the latest research progress of M. enterolobii in terms of ecological diffusion trends, pathogenic mechanisms, and green control, and explored the feasibility of integrating multidisciplinary technologies to construct an efficient and precise control system. The ultimate aim is to provide theoretical support and technical supports for green and sustainable development of global agriculture.

In November 2019, stem nematode was found on Codonopsis pilosula in Tanchang county, Gansu province, China. The population of stem nematode was identified on the basis of both molecular and morphological methods. The morphological and morphometric characteristics of this nematode population matched with Ditylenchus destructor Thorne, 1945. The sequences of rDNA-ITS and D2/D3 region of 28S-rRNA similarity with the D. destructor. The pathogenicity results revealed the symptom of dry rot on C. pilosula was caused by this nematode. To our knowledge, this is the first report that D. destructor on C. pilosula in China.

Introduction: Codonopsis pilosula root rot, caused by Fusarium oxysporum, has caused severe damage to the C. pilosula industry. Due to the unclear pathogenic mechanisms of F. oxysporum on C. pilosula, the effective implementation of control measures has been greatly restricted.Methods: An efficient protoplast preparation and genetic transformation system was established for F. oxysporum FO-1, enabling real-time tracking of fungal colonization in C. pilosula. Single-factor experiments were conducted to determine optimal conditions, followed by response surface methodology to further optimize enzymatic parameters. PEG-mediated transformation was performed to generate GFP-tagged strains for infection tracking.Results: Single-factor experiments identified the optimal conditions as 12-hour-old mycelia treated with 0.7 M NaCl and 20 mg/mL driselase at 28°C and 180 rpm for 4 h. Response surface methodology optimized parameters to 188.24 rpm, 4.51 h, and 27.5°C, yielding 1.44 × 10⁸ CFU/mL protoplasts, representing a 30-fold improvement over single-factor optimization. PEG-mediated transformation produced 11 GFP-tagged strains, with FO-GFP-7 retaining wild-type morphology, growth rate, and pathogenicity. Microscopic observation revealed infection dynamics: conidia aggregated at the rhizome by 2 days post-inoculation (dpi), followed by phloem colonization at 4 dpi and vascular invasion at 6 dpi. Wound inoculation at the rhizome accelerated infection, consistent with field disease patterns linked to soil microfauna-induced injuries.Discussion: This study provides a robust platform for investigating F. oxysporum pathogenicity in C. pilosula and offers guidance on protective measures to maintain rhizome integrity during cultivation.