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In order to clarify the optimal spore production and inoculation methods for watermelon stem blight (Stagonosporosis cucurbitaceae), and to lay a solid foundation for the screening and identification of watermelon stem blight resistant germplasm resources, this article focuses on the spore production and inoculation methods of watermelon stem blight. Two common spore production methods were compared from the aspects of spore production time, spore yield, and spore morphology of stem blight. It was found that the UV induced spore production method had shorter spore production time, more stable spore yield, normal spore morphology, and lower cost compared to the cucumber induced spore production method. Currently, it was the best spore production method for watermelon stem blight. Watermelon resistant and susceptible materials were also inoculated with the methods of inoculum cake in vitro, spot inoculation with live spore suspension, and spray inoculation with live spore suspension, respectively. The three inoculation methods were compared in terms of operation difficulty and accuracy, and it was found that each of the three inoculation methods had advantages and disadvantages. The difference of resistance and susceptibility of watermelon resistant and susceptible materials was obvious after inoculating in vitro with cake inoculation and in vivo spore suspension spot inoculation, while the susceptible materials showed moderate resistance after inoculating in vivo spore suspension spray inoculation. Through comparison, it was found that the in vitro bacterial cake inoculation method and the live spore suspension point grafting method were more reliable, but the disease grading standards of the in vitro bacterial cake inoculation method were not precise enough, and the small differences in resistance between materials were difficult to reflect; the live spore suspension point grafting method had high accuracy, but the operation was complex, time-consuming, and labor-intensive; in vivo spore suspension spray inoculation method was simple in operation, but it was difficult to accurately control the amount of inoculum, and the accuracy was general. Therefore, it was suggested that the live spore suspension spray inoculation method could be used for preliminary screening when identifying the resistance of large quantities of materials to Fusarium wilt, and the live spore suspension spot connection method could be used when identifying the resistance of a small number of materials to Fusarium wilt, and the isolated cake inoculation method could be used as an auxiliary method to further verify the inoculation results. [ABSTRACT FROM AUTHOR]

Objectives: Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. Results: This research demonstrates successful pilot-scale (100 L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5 × 109 spores mL−1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6–2.3 cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10 g molasses L−1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12 months of storage at room temperature. Conclusion: An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale. [ABSTRACT FROM AUTHOR]

In order to examine the pathogenicity of Alternaria dauci, the causal agent of carrot leaf blight, it is necessary to standardize sporulation conditions to ensure the optimal quantity and quality of spore inoculum. There-fore, in this study, the effects of the growth medium, aeration treatment, and UV treatment with 12-hr photo-period on the sporulation of A. dauci KACC42997 were investigated. A. dauci KACC42997 was pre-cultured for 7 days in a potato dextrose agar medium at 25°C in the dark condition. When the pre-culture, after removing aerial mycelia, was re-incubated for 5 days, with simultaneous aeration treatment and 12-hr cycle UV treatment at 20°C, the largest number of spores was produced. One hundred seventy isolates of A. dauci were isolated from major carrot growing regions such as Pyeongchang, Gumi and Jeju and tested for sporulation under the conditions established in this study. Except for 20 strains, all strains produced spores. Statistically significant differences in the extent of sporulation were found among local populations of A. dauci isolates, but no difference was observed in their pathogenicity on carrots.

Purpose The seemingly harmful effect of chemical insecticides to the environment, plants and human health, has led to prominent role of entomopathogenic fungi (ENPF) as biopesticide. However, the success of ENPF as biocontrol agent depends on their amenability to easy and cheap mass multiplication. Solid state fermentation (SSF) has emerged as an appropriate technology for management of agro-industrial residues and for their value addition. In this study, the potentiality of agricultural by-products for mass multiplication of Beauveria bassiana was determined. Methods Two sets of SSF experiments with agricultural by-products [tea leaf waste, wheat bran, husk of rice, pigeon pea and urad, and seed cake of jatropha and pongamia] as substrates; one with fortification of nutrients, while other with no nutrient addition were performed. The substrates (200 g) were inoculated with B. bassiana spore suspension (1 9 108 spores/ml) and incubated at 28 C for 14 days. After the incubation period, B. bassiana spore concentrations were enumerated. Results Maximum spore production was observed in rice husk, while jatropha seed cake showed least spore production. Addition of nutrient supplements in the substrates showed slight (pongamia seed cake) to significant (rice husk, tea leaf waste) increase in spore production. The results showed constant increase in spore production with increase in C/N ratio of the substrates. Conclusions Mass production processes directly influences the cost, shelf life, virulence, and field efficacy of fungal pathogens. The results from this investigation are expected to pave the way towards commercialization of B. bassiana by augmenting the research in fungal mass production.

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore‐forming bacteria. The endospore‐forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected. [ABSTRACT FROM AUTHOR]

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain‐specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one‐factor‐at‐a‐time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH‐control. Especially the pH‐control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%–90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH‐control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics. [ABSTRACT FROM AUTHOR]

Abstract The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore‐forming bacteria. The endospore‐forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.

Abstract Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain‐specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one‐factor‐at‐a‐time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH‐control. Especially the pH‐control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%–90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH‐control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.

Simple Summary: The tea shot-hole borer (TSHB) is an invasive ambrosia beetle that vectors several fungal plant pathogens that cause Fusarium branch dieback in avocado trees. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing and mitigating the spread of the TSHB. The formulated fungal products tested were BioCeres WP, BotaniGard WP, BotaniGard ES and Velifer ES. Acquisition of spores by adult beetles dipped in the product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the products in an in vivo avocado bark plug bioassay was also determined. Controls consisted of water only, BotaniGard ES and Velifer ES suspension supernatants with the spores removed. Production of Bb spores on beetles was assessed. Beetles exposed to Velifer ES and BotaniGard ES acquired significantly more spores than those exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6–8 days) compared to beetles treated with the other fungal products (10–11 days) and controls (12 days). Percentage mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES was the highest among all treatments, whereas it was lowest on cadavers of beetles dipped in BotaniGard ES. All fungal products in this laboratory study demonstrated potential for use in management of the TSHB, especially Velifer ES. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results. Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6–8 days) compared to beetles dipped in the other fungal products (10–11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results. [ABSTRACT FROM AUTHOR]

Bacillus amyloliquefaciens is a food spoilage spore‐forming bacterium. Its spores are useful for multiple biotechnological applications. Nevertheless, few reports are available regarding the achievement of a high cell density and good sporulation effectiveness under fermentation conditions. Therefore, the current study was designed to optimize a low‐cost fermentation medium allowing the highest sporulation yield by B. amyloliquefaciens strain BS13. Our data revealed that tryptone and starch were the best carbon and energy sources. In addition, two nitrogen sources namely, corn steep liquor (CSL) and yeast extract (YE), allowed a significant enhancement of spore production and they were both retained for further optimization. A combination of CaCl2, MgSO4, and MnSO4 showed a positive impact on spores' production. The composition of the optimized medium was (in g/L); tryptone 3, starch 15, CSL 13.5, YE 1.5, CaCl2 0.1, MgSO4·7H2O 0.012, and MnSO4·7H2O 0.0012. Such medium was further validated in a 400‐L fermentor. The spore yield by B. amyloliquefaciens strain BS13 was enhanced from 3.0 × 1010 spores/mL under flask culture conditions to 6.2 × 1010 spores/mL when cultures were performed on large scale. Therefore, strain BS13 spore preparation could be proposed as a promising probiotic and a biocontrol agent useful for plants, animals, and humans. [ABSTRACT FROM AUTHOR]

Abstract The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two‐step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one‐step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one‐step BI production is technically possible but at the expense of spore yield. The two‐step manufacturing process can realize almost 10‐fold higher spore yields.

Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6–8 days) compared to beetles dipped in the other fungal products (10–11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results.

Establishing diverse mycorrhizal fungal communities is considered important for forest recovery, yet mycorrhizae may have complex effects on tree growth depending on the composition of fungal species present. In an effort to understand the role of mycorrhizal fungi community in forest restoration in southern Costa Rica, we sampled the arbuscular mycorrhizal fungal (AMF) community across eight sites that were planted with the same species (Inga edulis, Erythrina poeppigiana, Terminalia amazonia, and Vochysia guatemalensis) but varied twofold to fourfold in overall tree growth rates. The AMF community was measured in multiple ways: as percent colonization of host tree roots, by DNA isolation of the fungal species associated with the roots, and through spore density, volume, and identity in both the wet and dry seasons. Consistent with prior tropical restoration research, the majority of fungal species belonged to the genus Glomus and genus Acaulospora, accounting for more than half of the species and relative abundance found on trees roots and over 95% of spore density across all sites. Greater AMF diversity correlated with lower soil organic matter, carbon, and nitrogen concentrations and longer durations of prior pasture use across sites. Contrary to previous literature findings, AMF species diversity and spore densities were inversely related to tree growth, which may have arisen from trees facultatively increasing their associations with AMF in lower soil fertility sites. Changes to AMF community composition also may have led to variation in disturbance susceptibility, host tree nutrient acquisition, and tree growth. These results highlight the potential importance of fungal-tree-soil interactions in forest recovery and suggest that fungal community dynamics could have important implications for tree growth in disturbed soils.

[Display omitted] • Two Trichoderma strains were isolated from the rhizospheric soil of medicinal plants. • Fermentation factors of lignocellulosic residues by Trichoderma strains was assessed. • Substrate components was a crucial factor affecting fermentation results. • T. afroharzianum showed pronounced cellulose degradation efficiency for corn straw. • Two Trichoderma strains would be useful for application in waste materials treatment. To achieve an economically viable substitutive strategy for lignocellulosic residues use, cellulose degradation by efficient Trichoderma strains become a key alternative. This study aimed to explore the effects of various cultural conditions on the degradation of different lignocellulosic substrates (corn straw and licorice residues) by Trichoderma longibrachiatum and Trichoderma afroharzianum in solid state fermentation. The strains were isolated from the rhizospheric soils of medicinal plants. In addition, the best fermentation combination was selected to evaluate the degradation results. This study confirmed that fermentation conditions and different components of corn straw and licorice residues were both crucial factors affecting the cellulase activity and spore production of two Trichoderma strains. A combination of T. afroharzianum degrading corn straw was selected for Response Surface Methodology optimization. The best fermentation conditions were fermentation time of 5.72 days, initial material liquid ratio of 1:4.58, initial pH of 6.32 and fermentation temperature of 28.8 ℃, with a filter paper cellulase activity of 0.91 U/mL. After continuous fermentation, the spore production reached 6.18 × 109 spores/g, the crude fiber degradation rate reached 23.5 % with the reducing sugar content maintained at 11 mg/g. These results highlight the great potential of T. afroharzianum degrading corn straw for organic amendment. [ABSTRACT FROM AUTHOR]

The mevalonate (MVA) pathway in eukaryotic organisms produces isoprenoids, sterols, ubiquinone, and dolichols. These molecules are vital for diverse cellular functions, ranging from signaling to membrane integrity, and from post-translational modification to energy homeostasis. However, information on the MVA pathway in Phytophthora species is limited. In this study, we identified the MVA pathway genes and reconstructed the complete pathway in Phytophthora sojae in silico. We characterized the function of the MVA pathway of P. sojae by treatment with enzyme inhibitor lovastatin, deletion of the geranylgeranyl diphosphate synthase gene (PsBTS1), and transcriptome profiling analysis. The MVA pathway is ubiquitously conserved in Phytophthora species. Under lovastatin treatment, mycelial growth, spore production, and virulence of P. sojae were inhibited but the zoospore encystment rate increased. Heterozygous mutants of PsBTS1 showed slow growth, abnormal colony characteristics, and mycelial morphology. Mutants showed decreased numbers of sporangia and oospores as well as reduced virulence. RNA sequencing analysis identified the essential genes in sporangia formation were influenced by the enzyme inhibitor lovastatin. Our findings elucidate the role of the MVA pathway in P. sojae and provide new insights into the molecular mechanisms underlying the development, reproduction, and virulence of P. sojae and possibly other oomycetes. Our results also provide potential chemical targets for management of plant Phytophthora diseases. [ABSTRACT FROM AUTHOR]

Spot blotch of wheat is an emerging threat that causes an annual yield loss of ~17%. The progression of the disease is believed to be dependent on the stage of growth. The response of seedlings to spot blotch infection remains unclear. Here, we have evaluated spot blotch resistance in wheat seedlings using (55) genotypes, covering a broad gene pool/pedigree. The germination of B. sorokiniana spores was independent of the growth stages of the plant. The initial number of lesions produced on seedlings was restricted to 10 in 47 genotypes. Such lesions did not increase in size and numbers in the inoculated seedlings. Conidiophores and conidia were not produced in the lesions. The reprogramming of SOD and MDA activity was reported after inoculation, and the maximum SOD activity was observed at 72 hours after inoculation. There was a negative correlation between SOD and MDA. The lignin content assessment and the activity of enzymes involved in the lignin biosynthesis pathway revealed their role in spot blotch resistance. This investigation is a step towards bridging the information gap by evaluating the germplasm of wheat and understanding the pattern of spot blotch resistance in seedlings and adult wheat. [ABSTRACT FROM AUTHOR]

The mevalonate (MVA) pathway in eukaryotic organisms produces isoprenoids, sterols, ubiquinone, and dolichols. These molecules are vital for diverse cellular functions, ranging from signaling to membrane integrity, and from post-translational modification to energy homeostasis. However, information on the MVA pathway in Phytophthora species is limited. In this study, we identified the MVA pathway genes and reconstructed the complete pathway in Phytophthora sojae in silico. We characterized the function of the MVA pathway of P. sojae by treatment with enzyme inhibitor lovastatin, deletion of the geranylgeranyl diphosphate synthase gene (PsBTS1), and transcriptome profiling analysis. The MVA pathway is ubiquitously conserved in Phytophthora species. Under lovastatin treatment, mycelial growth, spore production, and virulence of P. sojae were inhibited but the zoospore encystment rate increased. Heterozygous mutants of PsBTS1 showed slow growth, abnormal colony characteristics, and mycelial morphology. Mutants showed decreased numbers of sporangia and oospores as well as reduced virulence. RNA sequencing analysis identified the essential genes in sporangia formation were influenced by the enzyme inhibitor lovastatin. Our findings elucidate the role of the MVA pathway in P. sojae and provide new insights into the molecular mechanisms underlying the development, reproduction, and virulence of P. sojae and possibly other oomycetes. Our results also provide potential chemical targets for management of plant Phytophthora diseases.

Cucumber target leaf spot, caused by Corynespora cassiicola, is an emerging disease with a high incidence that causes severe damage to cucumbers on a global scale. Therefore, efforts need to be undertaken to limit the spread and infection of this pathogen, preferably by using environmentally friendly methods. In this study, the effects of temperature and moisture on the sporulation of C. cassiicola were investigated in vitro and in vivo. The novelty of our study refers to the observation of spore production and size as well as the revelation of a correlation between spore size and virulence. On potato dextrose agar (PDA) and cucumber−leaf extract agar (CEA), temperature played a critical role in spore production, which was strongly influenced by both temperature and moisture on detached leaves and cucumber seedlings. Maximum spore production was found at 30 °C on PDA and 25 °C on CEA, cucumber detached leaves and living plants. Lower spore productions were observed with a stepwise change of 5 °C. In addition, the largest spore production was found at 100% relative humidity (RH) in comparison to the other tested moisture. Moreover, moisture was found to be the most important factor affecting spore size, accounting for 83.09–84.86% of the total variance in length and 44.72–73.10% of the total variance in width. The longest−narrowest spores were formed at 100% RH, and the shortest−widest spores were formed at 75% RH. Furthermore, the result showed that larger spores of C. cassiicola were more virulent and small spores were avirulent. Our findings will contribute to the development of new strategies for the effective alleviation and control of cucumber target leaf spot.

Beauveria bassiana is an entomopathogenic fungus that is used for the biological control of different agricultural pest insects. B. bassiana is traditionally cultivated in submerged fermentation and solid-state fermentation systems to obtain secondary metabolites with antifungal activity and infective spores. This work presents the design and characterization of a new laboratory-scale biofilm bioreactor for the simultaneous production of oosporein and aerial conidia by B. bassiana PQ2. The reactor was built with materials available in a conventional laboratory. KLa was determined at different air flows (1.5-2.5 L/min) by two different methods in the liquid phase and in the exhaust gases. The obtained values showed that an air flow of 2.5 L/min is sufficient to ensure adequate aeration to produce aerial conidia and secondary metabolites by B. bassiana. Under the conditions studied, a concentration of 183 mg oosporein per liter and 1.24 x 109 spores per gram of support was obtained at 168 h of culture. These results indicate that the biofilm bioreactor represents a viable alternative for the production of products for biological control from B. bassiana. [ABSTRACT FROM AUTHOR]

Mucor circinelloides is a fungus that produces diverse spores throughout its life cycle. The sporangiospores, which are the most well-studied spores in this fungus, are asexual spores produced during aerial mycelial development. M. circinelloides has the potential to be used in diverse biotechnological applications. In this study, we propose rice (Oryza sativa) grains as an alternative substrate for inexpensive and large-scale sporangiospore production. The sporangiospores produced from rice and a yeast extract-peptone-glucose (YPG) medium exhibited similar protein and nucleic acid contents and phenotypes in terms of germination under different conditions and culture media, including similar virulence rates against the nematode Caenorhabditis elegans. Transgenic strains carrying self-replicative plasmids were sporulated on rice and showed plasmid stability similar to that of spores produced on the YPG medium. Approximately 20% of the spore population lost plasmids after the first passage on rice. These results reveal that rice is a suitable substrate for the mass production of sporangiospores in M. circinelloides. [ABSTRACT FROM AUTHOR]

Blue mold rot, caused by Penicillium expansum, is one of the most economically important post-harvest diseases of apple worldwide. The goals of this study were threefold: to evaluate the diversity of P. expansum isolates for mycelial growth, spore production and lesion diameter on apples; to estimate the effects of cold-storage facility conditions on P. expansum population structure; and to investigate the efficacy of three essential oils against P. expansum. The results showed that storage facilities applying fungicides and storing diverse fruit species selected for P. expansum isolates with a larger lesion diameter on apples. In addition, application of fungicides and diversification in stored fruit species significantly select for P. expansum isolates with higher levels of mycelial growth and spore production, respectively. Moreover, the diversity of host species of stored fruit accounted for 38% of the variability observed between storage facilities for the measured fitness parameters in P. expansum isolates and had a stronger effect on P. expansum population structure than fungicide treatment. Essential oils from Mentha pulegium and Syzygium aromaticum significantly decreased mycelial growth and spore production of P. expansum isolates in vitro. Mentha pulegium essential oil also significantly decreased the size of lesions associated with the blue mold rot of apples. Reducing the diversity of stored host species and applying M. pulegium essential oil may be useful in counter-selecting for aggressive P. expansum isolates and reducing losses due to blue mold rot during fruit storage.

The banana weevil (BW), Cosmopolites sordidus, is the main coleopteran pest of banana, causing up to 100% yield loss. In this study, we screened 20 isolates of entomopathogenic fungi (EPF) for the management of BW. In the lab, eight Beauveria bassiana isolates caused >50% mortality of the adult BW, whereas Metarhizium anisopliae and Isaria fumosorosea isolates were less pathogenic. B. bassiana isolates ICIPE 648, ICIPE 660 and ICIPE 273 were the most pathogenic, killing ≥80% of adult BW. B. bassiana isolate ICIPE 622 yielded the highest spores per BW cadaver (1.84 × 108 spores), followed by ICIPE 660, ICIPE 273 and ICIPE 648—1.17 × 108, 3.8 × 107 and 3.6 × 107 spores, respectively. ICIPE 273 had the shortest LT50 (5.3 days) followed by ICIPE 648 (9.8 days) and 660 (11.1 days). Similarly, the LC50 values for the three isolates were 5.18 × 107, 5.49 × 107 and 5.2 × 107 spores mL−1, respectively. In the field, ICIPE 273 and ICIPE 648 had the highest (31.3%) and lowest (20.8%) pathogenicity, respectively. This study indicates that the B. bassiana isolates ICIPE 273, ICIPE 648 and ICIPE 660 are potential candidates for the environmentally sustainable management of BW.

Beauveria bassiana is an entomopathogenic fungus that is used for the biological control of different agricultural pest insects. B. bassiana is traditionally cultivated in submerged fermentation and solid-state fermentation systems to obtain secondary metabolites with antifungal activity and infective spores. This work presents the design and characterization of a new laboratory-scale biofilm bioreactor for the simultaneous production of oosporein and aerial conidia by B. bassiana PQ2. The reactor was built with materials available in a conventional laboratory. KLa was determined at different air flows (1.5–2.5 L/min) by two different methods in the liquid phase and in the exhaust gases. The obtained values showed that an air flow of 2.5 L/min is sufficient to ensure adequate aeration to produce aerial conidia and secondary metabolites by B. bassiana. Under the conditions studied, a concentration of 183 mg oosporein per liter and 1.24 × 109 spores per gram of support was obtained at 168 h of culture. These results indicate that the biofilm bioreactor represents a viable alternative for the production of products for biological control from B. bassiana.

Horizontal gene transfer (HGT) has been identified as an important source of genomic innovation in fungi. However, how HGT drove the evolution of Alternaria alternata , a necrotrophic fungus which can be ubiquitously isolated from soil and various plants and decaying plant materials is largely known. In this study, we identified 12 protein-encoding genes that are likely acquired from lineages outside Pezizomycotina. Phylogenetic trees and approximately unbiased comparative topology tests strongly supported the evolutionary origin of these genes. According to their predicted functions, these HGT candidates are involved in nitrogen and carbohydrate metabolism. Especially, five genes of them were likely transferred as a physically linked cluster from Tremellales (Basidiomycota). Functionally knocking out the five-gene cluster in an A. alternata isolate causing citrus brown spot resulted in an 80% decrease in asexual spore production in the deletion mutant. We further knocked out each of these five genes in this cluster and the resultant single-gene deletion mutants exhibited a various degree of reduction in spore production. Except for conidiation, functions of these genes associated with vegetative growth, stress tolerance, and virulence are very limited. Our results provide new evidence that HGT has played important roles over the course of the evolution of filamentous fungi. [ABSTRACT FROM AUTHOR]

Spore-forming probiotic bacteria have received a wide and constantly increasing scientific and commercial interest. Among them, Bacillus species are the most studied and well-characterized Gram-positive bacteria. The use of bacilli as probiotic products is expanding especially rapidly due to their inherent ability to form endospores with unique survivability and tolerance to extreme environments and to produce a large number of valuable metabolites coupled with their bio-therapeutic potential demonstrating immune stimulation, antimicrobial activities and competitive exclusion. Ease of Bacillus spp. production and stability during processing and storage make them a suitable candidate for commercial manufacture of novel foods or dietary supplements for human and animal feeds for livestock, especially in the poultry and aquaculture industries. Therefore, the development of low-cost and competitive technologies for the production of spore-forming probiotic bacteria through understanding physiological peculiarities and mechanisms determining the growth and spore production by Bacillus spp. became necessary. This review summarizes the recent literature and our own data on the physiology of bacilli growth and spore production in the submerged and solid-state fermentation conditions, focusing on the common characteristics and unique properties of individual bacteria as well as on several approaches providing enhanced spore formation. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum (Fo) belongs to a group of soil-borne hyphomycetes that are taxonomically collated in the Fusarium oxysporum Species Complex (FOSC). Hitherto, those infecting bananas were placed in the forma specialis cubense (Foc). Recently, however, these genetically different Foc lineages were recognized as new Fusarium spp. placed in the Fusarium of Banana Complex (FOBC). A member of this complex F. odoratissimum II-5 that uniquely comprises the so-called Tropical Race 4 (TR4), is a major problem sweeping through production zones of Cavendish banana in several regions of the world. Because of this, there is an urgent need for a phenotyping method that allows the screening for resistance to TR4 of large numbers of banana genotypes. Most Fusarium species produce three types of spores: macroconidia, microconidia and the persistent chlamydospores that can contaminate soils for many years. Inoculum production has been an important bottleneck for efficient phenotyping due to the low or variable number of conidia and the elaborate laboratory procedures requiring specific infrastructure. Here, we report a rapid, simple and high-yielding spore production method for nine F. oxysporum formae speciales as well as the biocontrol species Fo 47 and Fo 618-12. For Fusarium spp. causing Fusarium wilt or Panama disease of banana, we used the protocol for four species comprising the recognized physiological races, including Tropical Race 4 (TR4). We subsequently tested the produced inoculum in comparative inoculation trials on banana plants to evaluate their efficiency. All assays resulted in typical symptoms within 10 weeks; significant differences in final disease ratings were observed, depending on inoculum concentration. Pouring inoculum directly onto banana plants showed the most consistent and reproducible results, as expressed in external wilting, internal discoloration and determined by real-time PCR assays on entire rhizomes. Moreover, this method allows the inoculation of 250 plants per hour by one individual thereby facilitating the phenotyping of large mutant and breeding populations. [ABSTRACT FROM AUTHOR]

The fungus Ramularia collo-cygni is the causal agent of the Ramularia leaf spot (RLS) disease of barley (Hordeum vulgare). Despite having been described over 100 years ago, our knowledge of the life cycle and epidemic development of R. collo-cygni is limited. To learn more about its epidemiology, inoculation techniques are among the major research needs since standardised inoculation procedures must be available to mimic natural infection of the pathogen under controlled conditions. The present study was conducted to obtain an insight into various factors affecting the growth and sporulation of R. collo-cygni in axenic culture. The fungus was cultured on four different media and subjected to two light regimens to determine conditions favourable for its growth and sporulation. We showed that conidial production can be enhanced by growing R. collo-cygni on barley straw agar, a medium that mimics the plant host, under white light, or on quarter-strength potato dextrose agar supplemented with V8 juice agar under ultraviolet-C light, depending on the fungal isolate. To verify the infection potential of the spores produced in axenic culture, an inoculation trial was performed. R. collo-cygni spore-inoculated barley plants accumulated fungal biomass, demonstrating successful infection. [ABSTRACT FROM AUTHOR]

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47 × 1010 spores g−1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82 × 1010 spores g−1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH2PO4, 1 g MgSO4·7H2O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105 × 1010 spores g−1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10 × 1011 spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture. [ABSTRACT FROM AUTHOR]

Neonectria ditissima, the causal agent of European canker in apples, conidia are available year-round in the Tasman region of New Zealand. Spore production and release in apple trees were quantified during natural and artificial rain events. Rain traps captured spores over 18 rain events (July-August 2017) from European canker lesions (excised and in planta) derived from 'Royal Gala' picking wound and rasp wound infections. Rain traps were monitored regularly to determine spore release over time. Differences in the number of conidia produced from lesions on the tree and excised lesions re-mounted in the tree were not significant. Lesions arising from picking wounds produced 7.2 times more spores on average than lesions arising from rasp wounds. Most spores were released within the first hour of rain and total spore release followed a logarithmic curve. Rain duration × volume of water determined the rate of conidia release. Excised lesions are suitable to study the effect of biotic and abiotic drivers on spore production. [ABSTRACT FROM AUTHOR]

In this study, the effects of several key factors to increase spore production by Bacillus subtilis subsp. KATMIRA 1933 were evaluated in shake flask experiments. In a synthetic medium, glucose concentration played a crucial role in the expression of bacilli sporulation capacity. In particular, maximum spore yield (2.3 × 10 spores/mL) was achieved at low glucose concentration (2 g/L), and further gradual increase of the carbon source content in the medium caused a decrease in sporulation capacity. Substitution of glucose with several inexpensive lignocellulosic materials was found to be a reasonable way to achieve high cell density and sporulation. Of the materials tested, milled mandarin peels at a concentration of 40 g/L served as the best growth substrate. In these conditions, bacilli secreted sufficient levels of glycosyl hydrolases, providing slow hydrolysis of the mandarin peel's polysaccharides to metabolizable sugars, providing the bacterial culture with an adequate carbon and energy source. Among nitrogen sources tested, peptone was found to favor spore production. Moreover, it was shown that cheese and cottage cheese whey usage, instead of distilled water, significantly increases spore formation. After optimization of the nutrient medium in the shake flask experiments, the technical feasibility of large-scale spore production by B. subtilis KATMIRA 1933 was confirmed in a laboratory fermenter. The spore yield (7 × 10 spores/mL) obtained using a bioreactor was higher than those previously reported. [ABSTRACT FROM AUTHOR]

The aim of this study was to gain a deeper fundamental knowledge on the physiology of B. amyloliquefaciens B-1895 for development of probiotic production technology through the submerged fermentation of renewable and inexpensive agro-industrial lignocellulosic biomass. For comparison, bacilli cultivation was performed in the synthetic medium with different carbon sources. The highest yield of spores (7.1 x 109/mL) was attained after 72 h of submerged cultivation of bacilli in the 0.4% glucose-containing medium. Substitution of glucose with various lignocellulosic materials at concentration of 40 g/L yielded 2.3-10.8 x 109 spores/mL. After subsequent optimization, the developed simple medium containing milled corn cobs as a growth substrate and casein hydrolysate at concentration of 40 mM N provided formation of as high as 2.8 x 1010 spore/mL in shake-flasks experiments and 2.5 x 1010 spores/mL in a laboratory fermenter enabling the large scale production of low-cost probiotic for their biotechnological application. The study underlines importance for the efficient sporulation of carbon source depletion at the end of the exponential growth phase. Moreover, it shows that an exploitation of lignocellulosic materials with an appropriate chemical composition is a reasonable way to achieve high cell density and sporulation since bacilli secrete sufficient levels of glycosyl hydrolases for substrate saccharification to ensure bacterial culture with carbon and energy source. [ABSTRACT FROM AUTHOR]

The formation of storage organs, such as spores and vesicles, is a central part of the life cycle of an arbuscular mycorrhizal fungus (AMF), but the conditions under which this occurs in AMF are not well understood. Here, quantity and distribution of storage organs formed by the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae within dead (excised) roots were characterised. 'Trap roots' (TR), separated from the growth substrate by a 30-μm mesh, supported hyphal growth and formation of storage organs of the AMF. Hyphae developed both inside and on the outside of the TR and also within air gaps of surrounding nylon mesh compartments, but formation of vesicles and spores was confined to the interior and to the surface of the TR. Up to 20 % of the TR length harboured newly formed storage organs, resulting in a number of about 60 per mg TR dry weight. The portion of TR length containing storage organs was greater in coarse (diameter >300 μm) than in thin (<150 μm) TR, irrespective of whether the TR were sourced from an AMF host or non-host plant. We conclude that the AMF's extraradical mycelium produces its storage organs within dead roots in preference to air space in the substrate. Dead roots may indirectly supply nutrients to AMF (once they have been mineralised) or represent a protected space for the fungal structures to develop. The experimental technique described here allows for the preparation of AMF spores and vesicles of F. mosseae free of any mineral substrate. [ABSTRACT FROM AUTHOR]

The CRISPRoff system was recently introduced as a programmable epigenetic memory writer that can be used to silence genes in human cells. The system makes use of a dead Cas9 protein (dCas9) that is fused with the ZNF10 KRAB, Dnmt3A, and Dnmt3L protein domains. The DNA methylation resulting from the CRISPRoff system can be removed by the CRISPRon system that consists of dCas9 fused to the catalytic domain of Tet1. Here, the CRISPRoff and CRISPRon systems were applied for the first time in a fungus. The CRISPRoff system resulted in an inactivation up to 100 % of the target genes flbA and GFP in Aspergillus niger. Phenotypes correlated with the degree of gene silencing in the transformants and were stable when going through a conidiation cycle, even when the CRISPRoff plasmid was removed from the flbA silenced strain. Introducing the CRISPRon system in a strain in which the CRISPRoff plasmid was removed fully reactivated flbA showing a phenotype similar to that of the wildtype. Together, the CRISPRoff and CRISPRon systems can be used to study gene function in A. niger., Competing Interests: Declaration of Competing Interest The authors do not have a declaration of interest., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Molds are important spoiler of food material and have potential of mycotoxin production. The adverse side effects of chemical preservatives have caused that research focused on use of naturally occurring compound, especially essential oils (EOs), for inhibition of mold growth. Essential oils are natural compounds containing terpenic mixtures that have antimicrobial and antioxidant properties. Zataria multifloro Boiss., a domestic aromatic plant of Iran, has different effect such as antispasmodic and antibacterial. Objective: In this study effect of Z. multiflora Boiss. Essential oil on growth and spore production by Aspergillus flavus and its morphological alteration action were investigated. Method: After extraction of essential oil with steam distillation method, it was analyzed by GC-MS. Evaluation of essential oil effect on growth and spore production carried out by agar dilution method. For SEM evaluation, mold cultured on PDA was fixed with osmium tetroxide and blocks of agar were coated with gold and observed by scanning electron microscope. Results: Effect of EO on growth and spore production by A. flavus was significant in all concentrations studied. MIC and MFC values were 400 and 1000 ppm, respectively. SEM analysis showed excessive sporulation of mold culture in absence of EO while sporulation of culture containing EO was very sparse. Morphological changes such as irregular surface with many folds and distortion of hyphae were seen in cultures treated with EO. Conclusion: Inhibitory effect of Z. multiflora Boiss. Essential oil demonstrated in this study candidate this EO as a substitution of chemical preservatives.