Mechanisms of Anaerobic Soil Disinfestation: Volatile Fatty Acids Reduce Viability of Athelia (Sclerotium) rolfsii Sclerotia in Acidic Soil Conditions and Have Limited Effects on Endemic Trichoderma spp.

Volatile fatty acids (VFAs), such as acetic and n-butyric acid, released during anaerobic decomposition of organic soil amendments during anaerobic soil disinfestation (ASD) likely play a role in soilborne plant pathogen inoculum suppression. However, research is limited on the direct effects of soil VFA exposure on fungal plant pathogen inoculum, effects on pathogen antagonists such as Trichoderma spp., and the role of soil microbial VFA metabolism on reducing exposure effects. The present study addresses these limitations through a series of studies evaluating the effects of VFA (acetic or n-butyric acid), VFA concentration (4, 8, or 16 mmol/kg soil), soil sterilization by autoclaving, and soil amendment on the viability of Athelia rolfsii (Sclerotium rolfsii) sclerotia post VFA exposure, and soil populations of Trichoderma spp. HCl and water-only controls were included. After 4-days exposure in an acidic, anaerobic environment, sclerotial viability, and colonization by culturable fungi or bacteria were assessed with standard procedures. Greenhouse experiments were similarly conducted to evaluate endemic soil populations of Trichoderma spp. following soil exposure to VFAs and Trichoderma spp. populations assessed with standard soil dilution plating onto semi-selective medium. Sclerotial germination was generally reduced by soil exposure to acetic (35.1% germination) or n-butyric (21.9% germination) acids compared to water (74.3% germination) and HCl (62.7% germination). Germination was reduced as VFA concentration increased from 4 to 8 and 16 mmol/kg (39.5, 29.1, and 16.9%, respectively). In amended soils, there was no difference in sclerotial germination compared to non-amended soils, but in the greenhouse experiment there was a Trichoderma spp. population increase of over 300% in amended soil [3.4 × 106 colony forming units (CFU)/g soil] compared to the non-amended soil (9.6 × 105 CFU/g soil). Soil autoclaving had no effect on sclerotial germination at low VFA concentrations, but sclerotial germination was reduced at higher VFA concentrations compared to non-autoclaved soil. Our results suggest that VFAs contribute to sclerotial mortality in strongly acidic soil environments, and mortality is influenced by VFA components and environment. Antifungal activity is less for acetic acid than for n-butyric, and less in non-sterile soil environments more typical of field conditions than in sterile laboratory conditions.

Compartmentalization of Melanin Biosynthetic Enzymes Contributes to Self-Defense against Intermediate Compound Scytalone in Botrytis cinerea

ABSTRACT In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite. IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.

Effect of Different Carbon and Nitrogen Sources on Sclerotium rolfsii sacc. Mycelial Growth and Sclerotial Development

In vitro studies were conducted on Potato Dextrose Agar using different carbon (C) and nitrogen (N) sources to evaluate their effects on the mycelial growth, and the sclerotial development of three Tunisian Sclerotium rolfsii Sacc. isolates. Radial growth was optimum on basal medium supplemented with ammonium chloride (0.48 gram of nitrogen per liter (g of N.L-1)) as N source but was restricted on L-Arginine and completely inhibited on ammonium acetate amended media (0.48 g N.L-1). Sclerotial initiation occurred from the 3rd to the 12th day of incubation for all tested isolates. Potassium nitrate was the most suitable N source for sclerotial formation whereas sclerotial development was completely inhibited on ammonium acetate amended medium. Optimal sclerotial germination was recorded using L-Arginine (78-80%) followed by L-Asparagine (46-94%) and ammonium chloride (46-88%) as N sources. Nevertheless, the lowest sclerotial germination rate was noted on sodium nitrate and ammonium acetate amended media. As for C sources (16 gram of carbon per liter (g of C.L-1)), optimal radial growth occurred using D-mannitol for Sr1 and Sr2 isolates and maltose for Sr3, but no mycelial growth was recorded using sodium citrate for all isolates. All C sources tested, except sodium citrate, were suitable for sclerotial formation, production, and germination. Mature sclerotia became brownish after 6 to 12 days of incubation and sclerotial production was highest using D-mannitol, maltose, and D-glucose, depending on isolates used, as C sources. Optimal germination of sclerotia was noted using D-glucose, D-mannitol and maltose for Sr1 isolate, maltose for Sr2 and D-glucose and maltose for Sr3. It was concluded that N and C sources are both important factors for the growth of S. rolfsii and its survival.

Rolled–crimped cereal rye residue suppresses white mold in no-till soybean and dry bean

White mold caused by the fungus, Sclerotinia sclerotiorum is a devastating disease of soybean (Glycine max) and other leguminous crops, including dry bean (Phaseolus vulgaris). Previous research has demonstrated that no-till planting soybean into rolled–crimped cereal rye residue can enhance weed management, improve soil health and reduce labor requirements in organic production. However, there are limited data on the effects of cereal rye residue on white mold suppression in no-till planted soybean and dry bean. Two field trials were conducted in 2016–2017 (Year 1) and repeated in 2017–2018 (Year 2) to evaluate the potential of cereal rye cover crop residue to suppress white mold in these crops. In each trial (soybean and dry bean), the experimental design was a randomized complete block with two treatments: (1) rolled–crimped cereal rye residue and (2) no cover crop control. Treatment effects on plant population, biomass and yield components varied between the main crops. Compared with the control treatment, cereal rye residue reduced the incidence of white mold in soybean in both years and in dry bean in Year 2. The reduction in white mold in cereal rye residue plots was due to a combination of (1) decreased sclerotial germination (no stipes formed) and (2) increased nonfunctional sclerotial germination defined here as sclerotia that germinated but produced stipes without the expanded cup where asci containing ascospores are formed. Weed density and biomass were lower in cereal rye residue plots in soybean and dry bean, except in Year 1 in soybean when weed biomass was low in both treatments. Our findings indicate that cereal rye residue could help organic and conventional farmers manage white mold in no-till planted soybean and dry bean. Germination of sclerotia resulting in nonfunctional apothecia could potentially exhaust soilborne inoculum in the upper soil profile and reduce infections in subsequent crops.

Effects of pH and Aeration on Sclerotium rolfsii sacc. Mycelial Growth, Sclerotial Production and Germination

Sclerotium rolfsii is one of the devastating soilborne fungus responsible for significant plant losses. The effects of pH and aeration on pathogen mycelial growth, sclerotial production and germination were investigated for three Tunisian isolates. Optimal mycelial growth occurred at pH 6 for Sr2 and Sr3 isolates and at pH 6-7 for Sr1. Dry mycelial growth was optimum at pH values ranging between 4 and 7. Sclerotial initiation started on the 3rd day of incubation at all pH values tested and mature sclerotia were formed after 6 to 12 days. Optimal sclerotial production was noted at pH 5. The dry weight of 100 sclerotia varied depending on isolates and pH and occurred at pH range 4-7. At pH 9, mycelial growth, sclerotial production and dry weight of 100 sclerotia were restricted. The optimum sclerotial germination, noted after 24 h of incubation, varied depending on isolates and pH and occurred at pH 4-9. Mycelial growth was optimum in aerated plates with a significant isolates x aeration treatments interaction. Sclerotial initiation occurred at the 3rd day of incubation and mature sclerotia were observed after 6-9 days. Sclerotial development was very slow in completely sealed plates and dark sclerotia were produced only after 15 days of incubation. The highest sclerotial yields were noted in aerated plates. The highest dry weight of 100 sclerotia for Sr1 isolate was recorded in ½ sealed, no sealed and completely sealed plates, while for Sr2, it was noted in ½ and ⅔ sealed plates. For Sr3, the maximum dry weight of 100 sclerotia was recorded in ½, ⅔ and completely sealed plates. Germination of S. rolfsii sclerotia, after 24 h of incubation, did not vary significantly depending on aeration treatments and ranged from 90 to 100% for all isolates.

Study on antifungal activity of silver/bentonite nanomaterials on soybean phytopathogenic fungi

In the present study, silver nanoparticles were synthesized by chemical reduction method route into the lamellar space of bentonite (Ag/CTS/Bentonite). Silver nitrate (AgNO3) was taken as a metal precursor, sodium borohydride (NaBH4) as a reducing agent, reduction of Ag+ ions and the subsequent formation of Ag nanoparticles, chitosan as a natural polymeric stabilizer and was sticked silver nanoparticles to the surface of bentonite particles, respectively. Manipulating the size and shape of Ag/CTS/Bentonite nanomaterial was characterized using transmission electron microscopy (TEM), spherical silver nanoparticles, as depicted by TEM, were found to have a wide particle size distribution from 5 nm to 90 nm. Meanwhile, the X-Ray flourescence (XRF) spectrum indicated the presence of silver on bentonite particles. Antifungal activity of the synthesized Ag/CTS/Bentonite nanomaterial was investigated against crop pathogenic fungi (Fusarium oxysporium (F. oxysporium) and Rhizoctonia solani (R. solani)) isolated from infected soybean plant in Bac Ninh province by the Plant Protection Research Institute. The assessment of fungicidal activity of the Ag/CTS/Bentonite nanomaterial showed that this product exhibited strong antifungal activity towards soybean pathogenic fungi. At highest nanosilver concentration of the Ag/CTS/Bentonite nanocomposite (400 ppm) sclerotial germination of F. oxysporum was almost inhibited, after 7 days the inhibition effect on sclerotial germination attained 66.70%. For the case of R. solani, after 2 days at 400 ppm silver nanoparticles concentration the inhibition effect on sclerotial germination attained 92.82%. The obtained results suggested that the synthesized Ag/CTS/Bentonite nanomaterial acts as an effective antifungal agent. Thus, it could be used in developing novel antifungal agents for potential applications in agriculture.

Anaerobic Soil Disinfestation Reduces Germination and Affects Colonization of Sclerotium rolfsii Sclerotia

Growth chamber and field studies were conducted with organic amendment mixtures of carbon (C) and nitrogen (N) at C:N ratios 10:1, 20:1, 30:1, and 40:1 and amendment rates of C at 2, 4, 6, and 8 mg/g of soil (C:N ratio 30:1) to evaluate anaerobic soil disinfestation (ASD) effects on germination and colonization of Sclerotium rolfsii. In the growth chamber, sclerotial germination was reduced in all ASD treatments regardless of C:N ratio (0.6 to 8.5% germination) or amendment rate (7.5 to 46%) as compared with nonamended controls (21 to 36% and 61 to 96%, respectively). ASD treatment increased Trichoderma spp. colonization of sclerotia, with consistently higher colonization in ASD treatments with amendment rates of C at 2 or 4 mg/g of soil (>87% colonization) compared with nonamended controls (<50% colonization). In the 2014 field study, sclerotial germination was reduced by 24 to 30% in ASD treatments when compared with the nonamended control. Sclerotial colonization by Trichoderma spp. was predominant; however, other potential mycoparasites (i.e., Aspergillus spp., Fusarium spp., zygomycetes, and other fungi) were present in the field study. Amendment C:N ratios in the range of 10:1 to 40:1 were equally effective in reducing sclerotial germination and enhancing colonization by potentially beneficial mycoparasites of sclerotia.