Conidial Germination Research Articles

The MYB-like protein MylA contributes to conidiogenesis and conidial germination in Aspergillus nidulans

Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.

Role of Flavohemoglobins in the Development and Aflatoxin Biosynthesis of Aspergillus flavus.

Aspergillus flavus is notorious for contaminating food with its secondary metabolite-highly carcinogenic aflatoxins. In this study, we found that exogenous nitric oxide (NO) donor could influence aflatoxin production in A. flavus. Flavohemoglobins (FHbs) are vital functional units in maintaining nitric oxide (NO) homeostasis and are crucial for normal cell function. To investigate whether endogenous NO changes affect aflatoxin biosynthesis, two FHbs, FHbA and FHbB, were identified in this study. FHbA was confirmed as the main protein to maintain NO homeostasis, as its absence led to a significant increase in intracellular NO levels and heightened sensitivity to SNP stress. Dramatically, FHbA deletion retarded aflatoxin production. In addition, FHbA played important roles in mycelial growth, conidial germination, and sclerotial development, and response to oxidative stress and high-temperature stress. Although FHbB did not significantly impact the cellular NO level, it was also involved in sclerotial development, aflatoxin synthesis, and stress response. Our findings provide a new perspective for studying the regulatory mechanism of the development and secondary mechanism in A. flavus.

Biological control of damping-off by Fusarium oxysporum and F. verticillioides on pine and oak seedlings using edible ectomycorrhizal fungi

The Mediterranean forest has an important ecological and economic role, being holm oak (Quercus ilex), Pyrenean oak (Quercus pyrenaica), umbrella pine (Pinus pinea) and Scot pine (Pinus sylvestris) some of its main tree species. The fungal damping-off disease caused by Fusarium seriously threatens the establishment of these forest species in nurseries and reforestation, requiring the search for environmentally friendly alternatives to control the disease. We have used different species of ectomycorrhizal fungi (EMF) as potential biological control agents (BCAs) effective against the disease: Lactarius sanguifluus, Tricholoma portentosum, Suillus luteus and Agaricus silvicola from Pinus-species, and Leccinum lepidum, Amanita rubescens and Xerocomus ferrugineus from Quercus-species. A direct in vitro confrontation was performed and conidial germination of Fusarium in contact with cell-free filtrates produced by EMF was studied. Le. lepidum was the most effective Quercus-fungus in vitro against F. oxysporum, reducing its growth up to 32 % and its conidial germination up to 87 %. S. luteus was the most effective Pinus-fungus in vitro against F. oxysporum and F. verticillioides, reducing in direct confrontation, reducing its growth up to 30 %. However, La. sanguifluus was the Pinus-fungus that inhibited conidial germination of both pathogens, up to 55 %. In planta trials were carried out with seeds of the four forest species growing on substrate colonized by Le. lepidum (in Q. ilex and Q. pyrenaica seeds, infected by F. oxysporum) or by La. sanguifluus (P. pinea and P. sylvestris seeds, infected by F. oxysporum or F. verticillioides). Only La. sanguifluus was effective in reducing disease caused by F. oxysporum (strain Fo4) on P. sylvestris seeds. Therefore, EMF may be a potential tool in the control of damping-off in forest species, requiring further research.

The new CFEM protein CgCsa required for Fe3+ homeostasis regulates the growth, development, and pathogenicity of Colletotrichum gloeosporioides

Secreted common fungal extracellular membrane (CFEM) domain proteins have been implicated in multiple biological functions in fungi. However, it is still largely unknown whether the ferric iron (Fe3+), as an important trace element, was involved with the biological function of CFEM proteins. In this study, a new CFEM protein CgCsa, with high expression levels at the early inoculation stage on peppers by Colletotrichum gloeosporioides was investigated. Deletion of the targeted gene CgCsa revealed multiple biological roles in hyphal growth restriction, highly reduced conidial yield, delayed conidial germination, abnormal appressorium with elongated bud tubes, and significantly reduced virulence of C. gloeosporioides. Moreover, in CgCsa mutants, the expression levels of four cell wall synthesis-related genes were downregulated, and cell membrane permeability and electrical conductivity were increased. Compared to the wild-type, the CgCsa mutants downregulated expressions of iron transport-related genes, in addition, its three-dimensional structure was capable binding with iron. Increase in the Fe3+ concentration in the culture medium partially recovered the functions of ΔCgCsa mutant. This is probably the first report to show the association between CgCsa and iron homeostasis in C. gloeosporioides. The results suggest an alternative pathway for controlling plant fungal diseases by deplete their trace elements.

Synergistic antifungal effects of the preservative ammonium propionate and medium chain fatty acids against dormant and germinating conidia, germ tubes and hyphae of Aspergillus chevalieri, a feed spoilage fungus

In feed, propionic acid is the weak organic acid of choice to prevent growth of spoilage fungi. For safe and easy industrial handling this antifungal agent is applied in the presence of neutralizing ammonium, which however has the disadvantage to negatively affect the efficacy of fungus-inhibiting properties of the formulation. In the present study we investigated the impact of medium chain fatty acids (MCFA) on the antifungal efficacy of an ammonium propionate formulation on dormant- and germinating conidia as well as germ tubes and hyphae of Aspergillus chevalieri, a xerophilic fungus predominant on moulded feed. Dormant conidia were not affected by 32 mM of ammonium propionate after a 28 h-treatment in demi water. Similar results were obtained with solely 0.52 mM MCFA. However, the combination of both components nearly eradicated formation of colonies from these conidia and was accompanied by distortion of the cellular structure as was visible with light- and transmission electron microscopy. Germination of conidia, characterised by swelling and germ tube formation, was significantly decreased in the presence of 16 mM ammonium propionate and 0.26 mM MCFA, while the latter component itself did not significantly decrease germination. We conclude that a combination of ammonium propionate and MCFA had a synergistic antifungal effect on dormant and germinating conidia.When the combination of ammonium propionate and MCFA was tested on hyphae for 30 min, we observed that cell death was significantly increased in comparison to components alone. Treatment of the hyphae with 16 mM of ammonium propionate caused aberrant mitochondria, as evidenced by irregularly shaped and enlarged mitochondria that contained electron-dense inclusions as observed by transmission electron microscopy. When the combination of ammonium propionate and MCFA was applied against the hyphae, more severe cell damage was observed, with signs of autophagy. Summarised, our results demonstrate synergistic antifungal effects of ammonium propionate and medium chain fatty acids on fungal survival structures, during their germination and after a short (sudden) treatment of growing cells. This is of potential importance for several areas of feed and food storage and shelf-life.

Prevalence of FRAC Group 11 Fungicide Resistance in Stemphylium vesicarium Isolates, but Not S. beticola Isolates, Causing Stemphylium Leaf Spot of Spinach (Spinacia oleracea).

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium, is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 μg/ml) than that of S. vesicarium (mean of 14.17 μg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium, including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola. The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola.

Isolation, characterization, and predatory activity of nematode-trapping fungus, Arthrobotrys oligospora PSGSS01

A research study was undertaken to find a fungus that could effectively trap and kill Meloidogyne spp., which are root-knot nematodes that cause significant crop loss worldwide. Soil samples were collected and processed appropriately for the isolation of nematode-trapping fungi. After a primary screening, a potential nematode-trapping fungus, Arthrobotrys oligospora (Orbilia oligospora) PSGSS01 was selected for further investigation. Its growth was tested under various conditions, including different media types, temperatures, and pH levels. Its predatory activity on Meloidogyne incognita was also assessed in vitro. The fungus exhibited septate double-celled conidiospores, trapping hyphae, and radiating and sparse mycelium. Optimal conditions for growth were found to be at 25 °C and a pH of 4.0 and 5.0 on corn meal agar. The best temperature and pH for conidiospore germination were 25°C − 37°C and 5.0, respectively. The isolate could trap 94% of second-stage juveniles of M. incognita, and 92% juvenile mortality was recorded in 120 h at 28°C. Therefore, the study concluded that the isolate A. oligospora (Orbilia oligospora) PSGSS01 could be used as a biological control agent against root-knot nematodes.

Identifying and Controlling Anthracnose Caused by Colletotrichum Taxa of Welsh Onion in Sanxing, Taiwan.

Leaves of Welsh onion (Allium fistulosum) are subject to various fungal diseases such as anthracnose (Colletotrichum species) and Stemphylium leaf blight (Stemphylium vesicarium). These diseases are the main biotic limitations to Welsh onion production in northern Taiwan. From 2018 to 2020, anthracnose symptoms were observed throughout Welsh onion fields in northern Taiwan, mainly the Sanxing area. In total, 33 strains of Colletotrichum species were isolated from diseased leaves, and major causative agents were identified based on a multilocus phylogenetic analysis using four genomic regions (act, tub2, gapdh, and internal transcribed spacer). Based on this phylogeny, Colletotrichum species causing anthracnose of Welsh onion were identified as C. spaethianum (C. spaethianum species complex) and C. circinans (C. dematium species complex) in the Sanxing area, northern Taiwan. To determine and compare the pathogenicity of each species, representative fungal strains of each species were inoculated on the cultivar 'Siao-Lyu' by spraying spore suspension onto the leaf surface. Welsh onion plants were susceptible to both species, but disease incidence and severity were higher in C. spaethianum. In total, 31 fungicides were tested to determine their efficacy in reducing mycelial growth and conidial germination of representative strains of C. spaethianum and C. circinans under laboratory conditions. Five fungicides-fluazinam, metiram, mancozeb, thiram, and dithianon-effectively reduced mycelial growth and spore germination in both C. spaethianum and C. circinans. In contrast, difenoconazole and trifloxystrobin + tebuconazole, which are commonly used in Welsh onion production in northern Taiwan, mainly the Sanxing area, were ineffective. These results serve as valuable insights for growers, enabling them to identify and address the emergence of anthracnose caused by C. spaethianum and C. circinans of Welsh onion, employing fungicides with diverse modes of action. The findings of this study support sustainable management of anthracnose in Sanxing, northern Taiwan, although further field tests of the fungicides are warranted.

Genome-Wide Identification of SNARE Family Genes and Functional Characterization of an R-SNARE Gene BbSEC22 in a Fungal Insect Pathogen Beauveria bassiana.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating cell membrane fusion and intracellular vesicular trafficking in eukaryotic cells, and have been well-documented to play critical roles in growth, development, and pathogenesis in the filamentous fungal plant pathogens. However, little is known about the contributions of SNAREs to the physiology and biocontrol potential in entomopathogenic filamentous fungi. Here, a genome-wide analysis of SNARE genes was performed taking advantage of the available whole genome sequence of Beauveria bassiana, a classical entomopathogenic fungus. Based on the compared genomic method, 22 genes encoding putative SNAREs were identified from the whole genome of B. bassiana, and were classified into four groups (7 Qa-, 4 Qb-, 6 Qc-, and 5 R-SNAREs) according to the conserved structural features of their encoding proteins. An R-SNARE encoding gene BbSEC22 was further functionally characterized by gene disruption and complementation. The BbSEC22 null mutant showed a fluffy appearance in mycelial growth and an obvious lag in conidial germination. The null mutant also exhibited significantly increased sensitivity to oxidative stress and cell wall perturbing agents and reduced the yield of conidia production by 43.1% compared with the wild-type strain. Moreover, disruption of BbSEC22 caused a significant decrease in conidial virulence to Spodoptera litura larvae. Overall, our results provide an overview of vesicle trafficking in B. bassiana and revealed that BbSec22 was a multifunctional protein associated with mycelial growth, sporulation, conidial germination, stress tolerance, and insecticidal virulence.

Gamma irradiation induced thermotolerance in Metarhizium spp. enhancing biocontrol of Nilapavarta lugens (Stål)

The Brown Planthopper (BPH), Nilaparvata lugens (Stål), is a devastating pest to rice crops in Southeast Asia, primarily due to its resistance to insecticides. As a result, the fungus Metarhizium spp. is used as an alternative control for BPH. However, their efficiency is limited due to delayed infectivity and sensitivity to environmental variables. In this study, two isolates of Metarhizium spp. MLUBN038 and MNMHN031, were induced for thermotolerance and biocontrol potential against Nilapavarta lugens (Stål). A multilocus phylogeny of four protein-coding genes including Beta-tubulin, rpb1, rpb2, and tef has identified the two isolates to be Metarhizium koreanum. Seventy-nine potential thermotolerant mutants were screened from the two isolates. These mutant isolates demonstrated significant tolerance to vegetative heat at 39 °C and enhanced resistance to wet heat stress at 45 °C after exposure for 60 h, outperforming the wild-type (WT) isolates. The mutant isolates also showed increased mycelial growth, conidial germination, and spore production compared to the WT isolates. Two mutant isolates, ML038_Mt 71 and MN031_Mt 46, exhibited the highest efficacy against BPH nymphs (4th-5th instars), resulting in a maximum mortality of 88.82 % and 83.55 %, respectively, compared to the WT (54.07 % and 47.54 %) after 14 days of inoculation in the greenhouse. These two highly virulent mutant isolates presented the lowest LT50 values of 2.88 and 3.72 days at a concentration of 1 × 109 conidia/mL. RAPD analysis revealed genetic diversity between the mutant and WT isolates, indicating distinct diversity profiles and supporting the successful generation of mutant variants. These findings suggest that the use of gamma irradiation and heat stress to induce mutations is crucial for the successful production of thermotolerant mutant isolates. Our results suggest that ML031_Mt 71 and MN031_Mt 46 have potential as effective biocontrol agents.

Biocontrol of strawberry Botrytis gray mold and prolong the fruit shelf-life by fumigant Trichoderma spp.

Objectives To screen high active volatile organic compounds (VOCs)-producing Trichoderma isolates against strawberry gray mold caused by Botrytis cinerea, and to explore their antagonistic mode of action against the pathogen. VOCs produced by nine Trichoderma isolates (Trichoderma atroviride T1 and T3; Trichoderma harzianum T2, T4 and T5; T6, T7, T8 and T9 identified as Trichoderma asperellum in this work) significantly inhibited the mycelial growth (13.9-63.0% reduction) and conidial germination (17.6-96.3% reduction) of B. cinerea, the highest inhibition percentage belonged to VOCs of T7; in a closed space, VOCs of T7 shared 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and detached leaves, respectively, prolonged the fruit shelf-life by 3 days in presence of B. cinerea, completely protected the leaves from B. cinerea infecting; volatile metabolites of T7 damaged the cell membrane permeability and integrity of B. cinerea, thereby inhibiting the mycelial growth and conidial germination. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the VOCs contain 23 potential compounds, and the majority of these compounds were categorised as alkenes, alcohols, and esters, including PEA and 6PP, which have been reported as substances produced by Trichoderma spp. T. asperellum T7 showed high biofumigant activity against mycelial growth especially conidial germination of B. cinerea and thus protected strawberry fruits and leaves from gray mold, which acted by damaging the pathogen's plasma membrane and resulting in cytoplasm leakage, was a potential biofumigant for controlling pre- and post-harvest strawberry gray mold.

Suppression of anthracnose disease by orsellinaldehyde isolated from the mushroom Coprinus comatus.

Anthracnose caused by Colletotrichum species is one of the most devastating diseases of fruits and crops. We isolated and identified an antifungal compound from the mushroom Coprinus comatus and investigated its inhibitory potential against anthracnose disease-causing fungi with the goal of discovering natural products that can suppress anthracnose-caused plant disease. The culture filtrate of C. comatus was subjected to a bioassay-guided isolation of antifungal compounds. The active compound was identified as orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde) based on mass spectroscopy and nuclear magnetic resonance analyses. Orsellinaldehyde displayed broad-spectrum inhibitory activity against different plant pathogenic fungi. Among the tested Colletotrichum species, it exhibited the lowest IC50 values on conidial germination and germ tube elongation of Colletotrichum orbiculare. The compound also showed remarkable inhibitory activity against Colletotrichum gloeosporiodes. The staining of Colletotrichum conidia with fluorescein diacetate and propidium iodide demonstrated that the compound is fungicidal. The postharvest in-vivo detached fruit assay indicated that orsellinaldehyde suppressed anthracnose lesion symptoms on mango and cucumber fruits caused by C. gloeosporioides and C. orbiculare, respectively. Orsellinaldehyde was identified as a potent antifungal compound from the culture filtrate of C. comatus. The inhibitory and fungicidal activities of orsellinaldehyde against different Colletotrichum species indicate its potential as a fungicide for protecting various fruits against anthracnose disease-causing fungi.

CsSNF1 kinase plays important roles in fungal development and virulence of the pepper anthracnose fungus Colletotrichum scovillei

AbstractPepper is a vegetable that is used and cultivated worldwide. The quality and production of pepper are reduced by fruit anthracnose disease caused by Colletotrichum scovillei. The highly conserved fungal sucrose non‐fermenting 1 (SNF1)‐related AMPK protein kinases are implicated in the pathogenicity and differentiation of several destructive foliar fungal pathogens; in addition, they affect fungal growth in the presence of glucose at different concentrations and under different environmental stresses. However, the function of SNF1 in C. scovillei is unclear. We functionally characterized SNF1 of C. scovillei using a targeted deletion mutant (ΔCssnf1) generated by homology‐dependent replacement. ΔCssnf1 was severely impaired in mycelial growth under glucose‐limited conditions; multiple defects in conidiation, conidial germination, appressorium formation, lipid degradation and vacuole formation were also observed. Compared to the wild type, ΔCssnf1 caused anthracnose disease of reduced severity on intact and wounded pepper fruits. These defects were recovered in the complemented strain (Cssnf1c). Taken together, our findings implicate CsSNF1 in the growth, development and virulence of C. scovillei.

Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

Physiological response of Metarhizium rileyi with linoleic acid supplementation

Metarhizium rileyi has a broad biocontrol spectrum but is highly sensitive to abiotic factors. A Colombian isolate M. rileyi Nm017 has shown notorious potential against Helicoverpa zea. However, it has a loss of up to 22 % of its conidial germination after drying, which limits its potential as a biocontrol agent and further commercialization. Conidial desiccation resistance can be enhanced by nutritional supplements, which promotes field adaptability and facilitates technological development as a biopesticide. In this study, the effect of culture medium supplemented with linoleic acid on desiccation tolerance in Nm017 conidia was evaluated. Results showed that using a 2 % linoleic acid-supplemented medium increased the relative germination after drying by 41 % compared to the control treatment, without affecting insecticidal activity on H. zea. Also, the fungus increased the synthesis of trehalose, glucose, and erythritol during drying, independently of linoleic acid use. Ultrastructural analyses of the cell wall-membrane showed a loss of thickness by 22 % and 25 %, in samples obtained from 2 % linoleic acid supplementation and the control, respectively. Regarding its morphological characteristics, conidia inner area from both treatments did not change after drying. However, conidia from the control had a 24 % decrease in length/width ratio, whereas there was no alteration in conidia from acid linoleic. The average value of dry conidia elasticity coefficient from linoleic acid treatment was 200 % above the control. Medium supplementation with linoleic acid is a promising fermentation strategy for obtaining more tolerant conidia without affecting production and biocontrol parameters, compatible solutes synthesis, or modifying its cell configuration.

Evaluation of fungicides for management of Botryosphaeriaceae associated with dieback in Australian walnut orchards

Dieback of fruiting spurs, stems and branches of walnut trees (Juglans regia L.), caused by Botryosphaeriaceae, is widespread in walnut orchards in Australia. Five species of Botryosphaeriaceae (Diplodia seriata, Dothiorella omnivora, Neofusicoccum macroclavatum, N. parvum, and Spencermartinsia viticola) were recovered from the Australian walnut orchards in a previous study, with D. seriata and N. parvum being the most prevalent. The present study evaluated inhibitory effects of ten fungicides on mycelium growth of those five species and on conidium germination of D. seriata and N. parvum. It investigated the preventative and curative efficacy of selected fungicides on disease incidence in glasshouse and field trials. In vitro experiments showed that nine of the fungicides reduced mycelium growth, and all ten inhibited conidium germination, but to varying extents. Tebuconazole, prochloraz manganese chloride, fluazinam, fludioxonil and pyraclostrobin were the most effective for inhibiting mycelium growth (EC50 < 0.14 µg a.i. mL-1), whereas pyraclostrobin, fluxapyroxad, fluopyram, penthiopyrad and tebuconazole were the most effective for inhibiting conidium germination (EC50 < 2.2 µg a.i. mL-1). In planta experiments with five fungicides confirmed that preventative treatments had greater efficacy than curative treatments. A field trial with four commercial fungicide formulations demonstrated that tebuconazole and tebuconazole + fluopyram provided protection of walnut trees for the longest period. The field trial also confirmed the efficacy of pyraclostrobin and the inhibitory effect of fluazinam. This study is the first in Australia to evaluate fungicides in different classes and with different modes of action for efficacy against Botryosphaeriaceae recovered from walnut orchards in Australia, and provides a wider selection of active ingredients for a fungicide rotation programme than that which is currently available to the Australian walnut industry.

Chitin Synthases in Cordyceps militaris: Genome-Wide Gene Identification, Evolutionary Insights, and Life Cycle Transcript Profiling

Chitin Synthases in Cordyceps militaris: Genome-Wide Gene Identification, Evolutionary Insights, and Life Cycle Transcript Profiling

Inhibitory Activities of Five Fungicides on Alternaria suffruticosae and Their Field Control Efficacy Against Tree Peony Black Spot.

Tree peony black spot (TPBS), mainly caused by Alternaria suffruticosae, is a common leaf disease on the ornamental peony, which poses a great threat to the flower buds in the current year and the flowering quality in the next year. However, there is only one fungicide registered for the control of this disease, difenoconazole. In order to avoid the severe problem of pathogen resistance caused by long-term use of difenoconazole, it is necessary to screen more chemical fungicides for the prevention and control of TPBS. In this study, the biological activities of flutolanil, phenamacril, pyraclostrobin, and boscalid on mycelial growth, conidial germination, germ tube elongation, and sporulation quantity of A. suffruticosae were determined, and the field control efficacy was tested to evaluate the preventive and therapeutic activities. Difenoconazole was used as a control simultaneously. The results showed that pyraclostrobin had the strongest inhibitory effects on the conidial germination, mycelium growth, germ tube elongation, and sporulation quantity, with the average EC50 values of 0.0517, 0.5343, 0.0008, and 0.8068 μg/ml, respectively. The inhibitory activity of flutolanil on the four developmental stages of A. suffruticosae was weaker than that of the other three fungicides. Compared with flutolanil, boscalid, the other succinate dehydrogenase inhibitor, had more strong inhibitory effects on the mycelial growth and sporulation quantity, with the average EC50 values of 3.8603 and 1.4760 μg/ml, respectively. Phenamacril had a moderate inhibitory level and had more inhibitory activity on conidial germination and germ tube elongation, with the average EC50 values of 31.5349 and 5.2597 μg/ml, respectively. All of the four fungicides had no significant effects on the shape of spores and germ tubes. The control fungicide difenoconazole had the strongest inhibitory activity on mycelial growth, and the average EC50 value was only 0.3297 μg/ml. However, its inhibitory activity on the other three growth stages was not high. In the field trials, pyraclostrobin had high control efficacy on TPBS even at low concentrations, reaching a minimum of 62.6293%, which was higher than that of difenoconazole. The other three fungicides had higher control efficacy at high concentrations but decreased significantly at low concentrations. Considering the dosage and control efficacy, pyraclostrobin was the first choice for the control of TPBS. Pyraclostrobin is the preferred alternative fungicide to difenoconazole for the prevention and control of TPBS in production.

Mentha spp. essential oils: toxicity to Alphitobius diaperinus, activity against poultry pathogenic bacteria, and Beauveria bassiana compatibility.

The botanical insecticide market is growing because of limitations placed on the use of certain synthetic chemical insecticides. In this sense, the lesser mealworm Alphitobius diaperius (Coleoptera: Tenebrionidae) is the main poultry pest. The insect causes weight loss and damage to the digestive system of poultry, and it is a vector and reservoir of pathogens. Consequently, this study explored the following hypotheses: (i) essential oils (EOs) derived from Mentha spp. are toxic to A. diaperius; (ii) these EOs are compatible with Beauveria bassiana, the natural enemy of the poultry pest, that parasite A. diaperinus; (iii) these EOs also exhibit activity against bacteria that are pathogenic to poultry. In topical applications and ingestion tests, EOs from Mentha arvensis, Mentha spicata, and Mentha piperita were toxic to A. diaperinus. Chromatographic analyses revealed that menthol is the predominant compound in M. arvensis and M. piperita, whereas carvone is the major compound in M. spicata. Both (-)- and (+)-menthol, along with (-)- and (+)-carvone, underwent testing with A. diaperinus. Nevertheless, their activity was not as potent as those of the EOs, suggesting a possible synergistic and/or additive effect. The EOs did not have any adverse effects on the conidial germination, vegetative growth, or conidia production per colony of the entomopathogenic fungus B. bassiana. Consequently, these EOs are compatible with this natural enemy. The EO extracted from M. spicata exhibited significant toxicity against Staphylococcus aureus (ATCC 25923), whereas the remaining EOs displayed moderate toxicity against this bacterium. The EOs derived from Mentha spp., as assessed in this study, hold promise for the development of botanical insecticides tailored for the control of A. diaperinus. These insecticides are selective in favor of the natural enemy B. bassiana and can also serve as effective sanitizers, thanks to their antibacterial properties.

The Effect of Humic Substances on the Colony Growth and Conidial Germination of Entomopathogenic Fungi from the Genus Metarhizium

The development of sustainable agricultural production involves the use of new plant protection products, with low toxicity to non-target organisms and high biodegradability. The aim of this study was to investigate the effect of commercially available preparations containing humic substances, in comparison with pure humic acids, on the growth and germination of spores of entomopathogenic fungi (EPFs) from the genus Metarhizium in vitro. AmiAGRA, HumiAGRA, AlgoHUM (recommended field dose) and pure humic acids extracted from peat, brown coal and spent mushroom substrate were added to Sabouraud’s culture substrate. Observation of the growth of the colonies of the tested species of EPFs was carried out every 5 days until day 20, measuring their diameter (mm). In the second stage of the experiment, the culture medium with the addition of preparations and pure humic acids was applied in a thin layer to the surface of glass slides and an aqueous solution with spores was introduced. Observation was carried out after 24 and 48 h, and the results obtained were expressed as percentages in relation to the control. The conducted research showed that on the 20th day of the culture (on average), preparations containing humic substances had a stimulating effect, while pure acids limited the growth of the colonies of the tested isolates slightly. The growth of M. anisopliae fungal colonies was most strongly stimulated by AlgoHUM, and M. flavoviride by HumiAGRA. The share of germinated spores after 48 h of contact with the substrate was higher than after 24 h, and more spores germinated on substrates with the addition of preparations containing humic substances than on pure humic acids.