Plant-Entomopathogenic Fungi Interaction: Recent Progress and Future Prospects on Endophytism-Mediated Growth Promotion and Biocontrol.

Entomopathogenic fungi, often acknowledged primarily for their insecticidal properties, fulfill diverse roles within ecosystems. These roles encompass endophytism, antagonism against plant diseases, promotion of the growth of plants, and inhabitation of the rhizosphere, occurring both naturally and upon artificial inoculation, as substantiated by a growing body of contemporary research. Numerous studies have highlighted the beneficial aspects of endophytic colonization. This review aims to systematically organize information concerning the direct (nutrient acquisition and production of phytohormones) and indirect (resistance induction, antibiotic and secondary metabolite production, siderophore production, and mitigation of abiotic and biotic stresses) implications of endophytic colonization. Furthermore, a thorough discussion of these mechanisms is provided. Several challenges, including isolation complexities, classification of novel strains, and the impact of terrestrial location, vegetation type, and anthropogenic reluctance to use fungal entomopathogens, have been recognized as hurdles. However, recent advancements in biotechnology within microbial research hold promising solutions to many of these challenges. Ultimately, the current constraints delineate potential future avenues for leveraging endophytic fungal entomopathogens as dual microbial control agents.

From the Photosynthesis to Hormone Biosynthesis in Plants.

Land plants produce glucose (C6H12O6) through photosynthesis by utilizing carbon dioxide (CO2), water (H2O), and light energy. Glucose can be stored in various polysaccharide forms for later use (e.g., sucrose in fruit, amylose in plastids), used to create cellulose, the primary structural component of cell walls, and immediately metabolized to generate cellular energy, adenosine triphosphate, through a series of respiratory pathways including glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Additionally, plants must metabolize glucose into amino acids, nucleotides, and various plant hormones, which are crucial for regulating many aspects of plant physiology. This review will summarize the biosynthesis of different plant hormones, such as auxin, salicylic acid, gibberellins, cytokinins, ethylene, and abscisic acid, in relation to glucose metabolism.

Evaluation of new strain (AAD16) of Beauveria bassiana recovered from Japanese rhinoceros beetle: Effects on three coleopteran insects.

A strain (AAD16) of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was isolated from field-collected Japanese rhinoceros beetle, Allomyrina dichotoma (L.) (Coleoptera: Scarabaeidae). Its virulence was compared with another strain (ARP14) recovered from a cadaver of Riptortus pedestris (F.) (Hemiptera: Alydidae) focusing on its effect on three coleopteran, i.e., Tenebrio molitor L., A. dichotoma, and Monochamus alternatus Hope. The LT50 value of T. molitor for two larval sizes, i.e., 16-18 and 22-24 mm, was 15.3 and 19.4% lower for strain AAD16 compared to strain ARP14, respectively. Furthermore, after 8 and 10 days of exposure, the mycosis rate of strain AAD16 was 1.3 and 1.2 times higher than that of strain ARP14 in the 16-18 and 22-24 mm larval sizes, respectively. The LT50 for M. alternatus larvae was 23.2% lower on strain AAD16 than on strain ARP14. In addition, the LT50 for M. alternatus adults was 47.1% lower for strain AAD16 compared to control. The mycosis rate of strain AAD16 on M. alternatus larvae was 1.8 higher than that of strain ARP14 after 120 hours of exposure. The strain AAD16 also showed higher larval mortality (90%) for A. dichotoma compared to strain ARP14 (45.0%) at 28 days after exposure. These results suggest that B. bassiana AAD16 can be a potential biological control agent against coleopteran pests.

Characterization of Three Fusarium spp. Causing Wilt Disease of Cannabis sativa L. in Korea.

In July 2021, wilting symptoms were observed in adult and seedling hemp (Cannabis sativa L. cv. Cherry Blossom) plants grown in a greenhouse. As the disease progressed, yellowing and wilting symptoms on the leaves developed, resulting in whole plant death. In seedling plants, typical damping-off symptoms were observed. To identify the pathogen, the roots of diseased plants were sampled, surface sterilized, and cultured on potato dextrose agar (PDA) media. From the culture, 4 different fungal isolates were recovered and purely cultured. Each fungal isolate showed distinct growth shapes and color development on malt extract agar, oatmeal agar, sabouraud dextrose agar, and PDA media. Microscopic observation and molecular identification using ribosomal DNA internal transcribed spacer sequencing identified them as 3 Fusarium spp. and 1 Thielaviopsis paradoxa. Additional sequencing of elongation factor 1-alpha and ß-tubulin regions of 3 Fusarium spp. revealed that 2 of them are Fusarium solani, and the other one is Fusarium proliferatum. To examine which isolate can act as a causal agent of wilt disease of hemp, each isolate was tested for their pathogenicity. In the pathogenicity test, F. solani AMCF1 and AMCF2, and F. proliferatum AMCF3, but not T. paradoxa AMCF4, were able to cause wilting disease in hemp seedlings. Therefore, we report that F. solani AMCF1 and AMCF2, and F. proliferatum AMCF3 as causal agents of Fusarium wilt of hemp plants. To our knowledge, this is the first report of the wilt disease of C. sativa L. caused by Fusarium spp. in Korea.

Isolation and Identification Antagonistic Bacterium Paenibacillus tianmuensis YM002 against Acidovorax citrulli.

In this study, we aimed to screen antagonistic microorganisms against Acidovorax citrulli, the causal agent of bacterial fruit blotch, which is known to induce sever diseases in cucurbit crops. From 240 bacterial strains isolated, only one unknown bacterial isolate, named YM002, showed significant antagonistic activity against A. citrulli KACC17909. Further experiments revealed that YM002 shows antagonistic activity against all tested A. citrulli strains, including KACC17000, KACC17001 and KACC17005, to different degrees. The phylogenetic analysis of 16S rRNA sequences identified YM002 as Paenibacillus tianmuensis. Importantly, pretreatment of cucumber (Cucumis sativus) leaves with YM002 enhanced disease resistance as observed by significantly reduced necrotic symptom development and bacterial growth. YM002-induced resistance accompanied by enhanced expression of defense-related genes, such as PAL1, PR1-1a and CTR1. Importantly, culture filtrate of YM002 significantly suppressed biofilm formation and swimming motility of A. citrulli, which is indispensable for its full virulence. In addition to its antagonistic activity, YM002 showed a various plant growth promotion (PGP)-related traits, such as production of ammonia production, amylase production, ACC deaminase production, inodole-3-acetic acid production, extracellular protease production, siderophore production, and zinc solubilization activities. Indeed, treatment of cucumber roots with YM002 significantly enhanced plant growth parameters, such as fresh and dry weight of leaves or roots. This study suggests the potential of YM002 as an effective PGPR with biological control activity against Acidovorax citrulli in cucumber plants.

Profiling Cannabinoid Contents and Expression Levels of Corresponding Biosynthetic Genes in Commercial Cannabis (Cannabis sativa L.) Cultivars.

Cannabis (Cannabis sativa L.) is widely cultivated and studied for its psychoactive and medicinal properties. As the major cannabinoids are present in acidic forms in Cannabis plants, non-enzymatic processes, such as decarboxylation, are crucial for their conversion to neutral active cannabinoid forms. Herein, we detected the levels of cannabidivarin (CBDV), cannabidiol (CBD), cannabichromene (CBC), and Δ9-tetrahydrocannabinol (Δ9-THC) in the leaves and vegetative shoots of five commercial Cannabis cultivars using a combination of relatively simple extraction, decarboxylation, and high-performance liquid chromatography analyses. The CBDV, CBC, and Δ9-THC levels were 6.3-114.9, 34.4-187.2, and 57.6-407.4 µg/g, respectively, and the CBD levels were the highest, ranging between 1.2-8.9 µg/g in leaf and vegetative shoot tissues of Cannabis cultivars. Additionally, correlations were observed between cannabinoid accumulation and transcription levels of genes encoding key enzymes for cannabinoid biosynthesis, including CsCBGAS, CsCBDAS, CsCBCAS, and CsTHCAS. These data suggest that the high accumulation of cannabinoids, such as CBC, Δ9-THC, and CBD, might be derived from the transcriptional regulation of CsCBGAS and CsCBDAS in Cannabis plants.

Biological Control Activity of Plant Growth Promoting Rhizobacteria Burkholderia contaminans AY001 against Tomato Fusarium Wilt and Bacterial Speck Diseases.

Plant growth promoting rhizobacteria (PGPR) is not only enhancing plant growth, but also inducing resistance against a broad range of pathogens, thus providing effective strategies to substitute chemical products. In this study, Burkholderia contaminans AY001 (AY001) is isolated based on its broad-spectrum antifungal activity. AY001 not only inhibited fungal pathogen growth in dual culture and culture filtrate assays, but also showed various PGPR traits, such as nitrogen fixation, phosphate solubilization, extracellular protease production, zinc solubilization and indole-3-acetic acid (IAA) biosynthesis activities. Indeed, AY001 treatment significantly enhanced growth of tomato plants and enhanced resistance against two distinct pathogens, F. oxysporum f.sp. lycopersici and Pseudomonas syringae pv. tomato. Real-time qPCR analyses revealed that AY001 treatment induced jasmonic acid/ethylene-dependent defense-related gene expression, suggesting its Induced Systemic Resistance (ISR)-eliciting activity. Gas chromatography-mass spectrometry (GC-MS) analysis of culture filtrate of AY001 revealed production of antimicrobial compounds, including di(2-ethylhexyl) phthalate and pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl). Taken together, our newly isolated AY001 showed promising PGPR and ISR activities in tomato plants, suggesting its potential use as a biofertilizer and biocontrol agent.

DNA-Based Method for Traceability and Authentication of Apis cerana and A. dorsata Honey (Hymenoptera: Apidae), Using the NADH dehydrogenase 2 Gene.

Honey is a widely used natural product and the price of honey from Apis cerana (ACH) and A. dorsata (ADH) is several times more expensive than the one from A. mellifera (AMH), thus there are increasing fraud issues reported in the market by mislabeling or mixing honeys with different entomological origins. In this study, three species-specific primers, targeting the NADH dehydrogenase 2 (ND2) region of honeybee mitochondrial DNA, were designed and tested to distinguish the entomological origin of ACH, ADH, and AMH. Molecular analysis showed that each primer set can specifically detect the ND2 region from the targeted honeybee DNA, but not from the others. The amplicon size for A. cerana, A. dorsata and A. mellifera were 224, 302, and 377 bp, respectively. Importantly, each primer set also specifically produced amplicons with expected size from the DNA prepared from honey samples with different entomological origins. The PCR adulteration test allowed detection of 1% of AMH in the mixture with either ACH or ADH. Furthermore, real-time PCR and melting curve analysis indicated the possible discrimination of origin of honey samples. Therefore, we provide the newly developed PCR-based method that can be used to determine the entomological origin of the three kinds of honey.

Biocontrol Activity of Aspergillus terreus ANU-301 against Two Distinct Plant Diseases, Tomato Fusarium Wilt and Potato Soft Rot.

To screen antagonistic fungi against plant pathogens, dual culture assay (DCA) and culture filtrate assay (CFA) were performed with unknown soil-born fungi. Among the different fungi isolated and screened from the soil, fungal isolate ANU-301 successfully inhibited growth of different plant pathogenic fungi, Colletotrichum acutatum, Alternaria alternata, and Fusarium oxysporum, in DCA and CFA. Morphological characteristics and rDNA internal transcribed spacer sequence analysis identified ANU-301 as Aspergillus terreus. Inoculation of tomato plants with Fusarium oxysporum f. sp. lycopersici (FOL) induced severe wilting symptom; however, co-inoculation with ANU-301 significantly enhanced resistance of tomato plants against FOL. In addition, culture filtrate (CF) of ANU-301 not only showed bacterial growth inhibition activity against Dickeya chrysanthemi (Dc), but also demonstrated protective effect in potato tuber against soft rot disease. Gas chromatography-tandem mass spectrometry analysis of CF of ANU-301 identified 2,4-bis(1-methyl-1-phenylethyl)-phenol (MPP) as the most abundant compound. MPP inhibited growth of Dc, but not of FOL, in a dose-dependent manner, and protected potato tuber from the soft rot disease induced by Dc. In conclusion, Aspergillus terreus ANU-301 could be used and further tested as a potential biological control agent.

AtTIP2;2 facilitates resistance to zinc toxicity via promoting zinc immobilization in the root and limiting root-to-shoot zinc translocation in Arabidopsis thaliana.

Zinc (Zn) is an essential micronutrient for plants. However, excess Zn is toxic to non-accumulating plants like Arabidopsis thaliana. To cope with Zn toxicity, non-accumulating plants need to keep excess Zn in the less sensitive root tissues and restrict its translocation to the vulnerable shoot tissues, a process referred to as Zn immobilization in the root. However, the mechanism underlying Zn immobilization is not fully understood. In Arabidopsis, sequestration of excess Zn to the vacuole of root cells is crucial for Zn immobilization, facilitated by distinct tonoplast-localized transporters. As some members of the aquaporin superfamily have been implicated in transporting metal ions besides polar but non-charged small molecules, we tested whether Arabidopsis thaliana tonoplast intrinsic proteins (AtTIPs) could be involved in Zn immobilization and resistance. We found that AtTIP2;2 is involved in retaining excess Zn in the root, limiting its translocation to the shoot, and facilitating its accumulation in the leaf trichome. Furthermore, when expressed in yeast, the tonoplast-localized AtTIP2;2 renders glutathione (GSH)-dependent Zn resistance to yeast cells, suggesting that AtTIP2;2 facilitates the across-tonoplast transport of GSH-Zn complexes. Our findings provide new insights into aquaporins' roles in heavy metal resistance and detoxification in plants.