Microsclerotia Production Research Articles

The role of VdSti1 in Verticillium dahliae: insights into pathogenicity and stress responses.

Sti1/Hop, a stress-induced co-chaperone protein, serves as a crucial link between Hsp70 and Hsp90 during cellular stress responses. Despite its importance in stress defense mechanisms, the biological role of Sti1 in Verticillium dahliae, a destructive fungal pathogen, remains largely unexplored. This study focused on identifying and characterizing Sti1 homologues in V. dahliae by comparing them to those found in Saccharomyces cerevisiae. The results indicated that the VdSti1-deficient mutant displayed increased sensitivity to drugs targeting the ergosterol synthesis pathway, leading to a notable inhibition of ergosterol biosynthesis. Moreover, the mutant exhibited reduced production of microsclerotia and melanin, accompanied by decreased expression of microsclerotia and melanin-related genes VDH1, Vayg1, and VaflM. Additionally, the mutant's conidia showed more severe damage under heat shock conditions and displayed growth defects under various stressors such as temperature, SDS, and CR stress, as well as increased sensitivity to H2O2, while osmotic stress did not impact its growth. Importantly, the VdSti1-deficient mutant demonstrated significantly diminished pathogenicity compared to the wild-type strain. This study sheds light on the functional conservation and divergence of Sti1 homologues in fungal biology and underscores the critical role of VdSti1 in microsclerotia development, stress response, and pathogenicity of V. dahliae.

Identification and virulence test of a new pathogen that causes verticillium striping on rapeseed in northwestern China

Five stems of rapeseed with abundant black microsclerotia were collected from Huangyuan County of Qinghai Province, China, and fungal isolates were obtained from the stems. They were identified based on morphology, molecular features and specific PCR detection. The results showed that the 10 fungal isolates belonged to Verticillium longisporum lineage A1/D3. One of the 10 isolates (HW7-1) was tested for virulence on three species of rapeseed, including B. napus Zhongshuang 9, B. rapa Qingyou 9 and B. juncea Tayou 2 by conidia inoculation of HW7-1 on roots of young seedlings. Control seedlings were inoculated with V. dahliae conidia or water alone. The seedlings of these treatments were transplanted in culture mix and incubated in a growth chamber (20 ​°C). Results suggested that the control seedlings of three cultivars appeared quite healthy, while the seedlings inoculated with HW7-1 turned yellowing leaves, seedling stunting or even death after 22 days post-inoculation. V. longisporum was re-isolated from he yellow leaves, thus fulfilling Koch's postulates. Moreover, compared to the control treatments, inoculation with HW7-1 caused flowering delay and seed yield reduction on Tayou 2 with production of microsclerotia on the stems. To our knowledge, this is the first report of V. longisporum lineage A1/D3 on rapeseed in northwestern China.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

Pathogenicity of microsclerotia from Metarhizium robertsii against Aedes aegypti larvae and antimicrobial peptides expression by mosquitoes during fungal-host interaction

Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii, i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae.

Identification of Bacillus velezensis SBB and Its Antifungal Effects against Verticillium dahliae.

Traditional control methods have drawbacks in controlling Verticillium wilt diseases caused by Verticillium dahliae Kleb.; therefore, an efficient and environmentally friendly strategy for disease control must be identified and the mechanisms determined. In this study, a soil-isolated strain SBB was identified as Bacillus velezensis based on 16S rRNA, gyrA, and gyrB gene sequences. In vitro, strain SBB had excellent inhibitory effects on V. dahliae, with the highest inhibition rate of 70.94%. Moreover, strain SBB inhibited production of the conidia of V. dahliae and suppressed the production of microsclerotia and melanin. Through gas chromatograph–mass spectrometer analysis, nine compounds were detected from the volatile organic compounds produced by SBB, among which 2-nonanol, 2-heptanone, 6-methyl-2-heptanone, and 2-nonanone could completely inhibit V. dahliae growth. Strain SBB produced cellulase, amylase, protease, and siderophore. During inhibitory action on V. dahliae, strain SBB showed upregulated expression of genes encoding non-volatile inhibitory metabolites, including difficidin, bacilysin, and bacillaene, at 1.923-, 1.848-, and 1.448-fold higher, respectively. Thus, our study proved that strain SBB had an efficient antagonistic effect on V. dahliae, suggesting strain SBB can be used as a potential biological control agent against Verticillium wilt.

Production of microsclerotia by Trichoderma asperellum through submerged liquid fermentation using low-cost nitrogen and carbon sources

The aim of this work is to optimize the nutritional conditions using low-cost carbon and nitrogen sources for microsclerotia (MS) production of two Brazilian Trichoderma asperellum strains (BRM-29576 and BRM-29104). A fractional factorial design was used to investigate five variables: carbon source, carbon concentration, nitrogen source, C:N ratio and fungal strain. In this study, the optimal nutritional conditions for improving MS yield were achieved using sucrose and autolyzed yeast, carbon concentration at 20 g L−1 and C:N ratio of 10:1 for the BRM-29104 strain, which outperformed the other strain and yielded up to 2.5 (±0.9) × 104 MS mL−1 within 7 days of fermentation. Sucrose and autolyzed yeast are two economically available carbon and nitrogen sources in Brazil that are suitable for MS production by T. asperellum BRM-29104. Vacuum filtration of MS biomass through nylon filter (200 mesh/74 μm) increased MS concentration, resulting in up to 1.8 (±0.2) × 105 MS g−1. Our results support a cost-efficient liquid medium to achieve high MS yields by strain BRM-29104 in combination with an easy method to harvest and concentrate MS from culture broth for further formulation development.

Clonostachys rosea: Production by Submerged Culture and Bioactivity Against Sclerotinia sclerotiorum and Bemisia tabaci.

Among the prospective biocontrol agents, the saprophytic filamentous fungus Clonostachys rosea is an excellent necrotrophic mycoparasite of numerous plant pathogenic fungi. However, its commercial development has been hampered by mass production difficulties during solid-state fermentation. Conversely, the submerged liquid fermentation shortens the cultivation time while increasing yields of fungal propagules. However, this method has been overlooked for C. rosea. In this work, we investigated the impact of liquid pre-culture inoculum on the spore production by the two-stage fermentation process using rice grains in comparison to the traditional solid-state fermentation. In parallel, we studied the submerged cultivation of C. rosea by manipulating carbon-to-nitrogen (C:N) ratio and nitrogen source, with the further optimization of spore production in a benchtop bioreactor. Additional bioassays included assessing the bioactivity of water-dispersible microgranules (that contained a submerged conidia) against the whitefly (Bemisia tabaci biotype B) and Sclerotinia sclerotiorum (causal agent of the white mold). Our results showed a maximum concentration of 1.1 × 109 conidia/g-dry-matter after 7 days of cultivation by two-stage fermentation process. The liquid fermentation yielded 1.4 × 109 submerged conidia/ml after 7 days using a medium with a 50:1 C:N ratio, and it also induced the production of microsclerotia (MS) up to 1.35 × 104/ml within 6 days with 10:1 C:N ratio; both media were supplemented with dextrose monohydrate and soybean meal. The fermentation batches carried out in a benchtop bioreactor with medium 50:1 C:N ratio and amended with soybean meal rendered a production peak on the fourth day, corresponding to 1.11 × 109 conidia/ml and 4.35 × 108 colony forming units (CFU)/ml. Following air-drying, the conidia production from air-dried microgranules of C. rosea biomass was estimated at 3.4 × 1010 conidia/g of formulated product upon re-hydration for 7 days. Both submerged conidia and MS of C. rosea inhibited 100% germination of S. sclerotiorum sclerotia by direct parasitism. The air-dried submerged conidia exhibited a suppressive activity on sclerotia (88% mycoparasitism) and early whitefly nymphs (76.2% mortality) that rendered LC50 values of 3.2 × 104 CFU/g soil and 1.5 × 107 CFU/ml, respectively. Therefore, the submerged liquid culture of C. rosea may offer a feasible and cost-effective method for its large-scale production, alleviating critical constraints to their commercial use while providing an additional tool for management of B. tabaci and S. sclerotiorum.

Production of Microsclerotia by Metarhizium sp., and Factors Affecting Their Survival, Germination, and Conidial Yield.

Microsclerotia (MS) produced by some species of Metarhizium can be used as active ingredients in mycoinsecticides for the control of soil-dwelling stages of geophilic pests. In this study, the MS production potential of two Metarhizium brunneum strains and one M. robertsii strain was evaluated. The three strains were able to produce MS in liquid fermentation, yielding between 4.0 × 106 (M. robertsii EAMa 01/158-Su strain) and 1.0 × 107 (M. brunneum EAMa 01/58-Su strain) infective propagules (CFU) per gram of MS. The EAMa 01/58-Su strain was selected for further investigation into the effects of key abiotic factors on their survival and conidial yield. The MS were demonstrated to be stable at different storage temperatures (−80, −18, and 4 °C), with a shelf-life up to one year. The best temperature for MS storage was −80 °C, ensuring good viability of MS for up to one year (4.9 × 1010 CFU/g MS). Moreover, soil texture significantly affected CFU production by MS; sandy soils were the best driver of infective propagule production. Finally, the best combination of soil temperature and humidity for MS germination was 22.7 °C and 7.3% (wt./wt.), with no significant effect of UV-B exposure time on MS viability. These results provide key insights into the handling and storage of MS, and for decision making on MS dosage and timing of application.

Verticillium diseases of vegetable crops in Brazil: Host range, microsclerotia production, molecular haplotype network, and pathogen species determination

AbstractRecent outbreaks of Verticillium wilt diseases in various vegetable crops have been reported in Brazil. This fact was our initial stimulus to carry out a nationwide survey aiming to determine their causal agent(s). Thus far, Verticillium dahliae has been reported as the predominant species based solely on morphological traits. As other Verticillium species can be associated with wilt diseases, we characterized a collection of 80 isolates, collected across 10 agricultural Brazilian regions, by combining morphological, biological, and molecular traits. A multilocus approach was employed for identification of Verticillium species with information from three genomic regions (ribosomal internal transcribed spacer region, glyceraldehyde‐3‐phosphate dehydrogenase, and actin genes). Only 21 out of the 80 isolates were unable to produce microsclerotia in culture. The analyses of all genomic regions indicated V. dahliae as the sole species associated with vascular wilt of distinct hosts, including major solanaceous vegetables and other hosts such as strawberry, okra, and cacao. Pathogenicity tests confirmed the infection by the V. dahliae isolates and the development of typical disease symptoms on their original hosts. This is the first nationwide characterization of Verticillium isolates associated with major vegetable crops in Neotropical areas. This provides valuable information to design sound management strategies for these diseases, mainly for establishing efficient rotation systems and for the development of resistant cultivars.

First report of Verticillium wilt and mortality of Ailanthus altissima caused by Verticillium dahliae and V. albo-atrum sensu lato in Spain.

Ailanthus altissima (Mill.) swingle is a highly invasive tree that has become established worldwide, especially in the Mediterranean Basin because of its good drought resistance. Ailanthus altissima is included in the list of Invasive Alien Species of the EU, so measures for eradication and management are required. Assessment for potential biological control agents is of great interest to manage this invasive tree in natural ecosystems. Verticillium dahliae Kleb. and Verticillium nonalfalfae Inderb. et al. (formerly V. albo-atrum Reinke & Berthold) have been reported as the causal agents of Verticillium wilt and mortality of ailanthus (Shall and Davis 2009; Rebbeck et al., 2013; Snyder et al., 2013; Brooks et al. 2020). Ailanthus trees with Verticillium wilt symptoms (wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality) were detected for the first time in 2007 in Celrà (42.040466N, 2.864436E) (Catalonia, Northeastern Spain), then spread to neighboring ailanthus populations. In 2018, ailanthus trees in a 570 km2 area in Catalonia were surveyed for disease symptoms. The incidence of wilt disease in ailanthus trees in forest ecosystems ranged from 50 to 90%, and the severity, 60 to 92%. One hundred and fifty branch samples showing wilt symptoms were collected and disinfected by immersion in 1% sodium hypochlorite for 2 min, then cut into 5mm pieces. These were placed onto PDA plates and incubated at 22.5 °C and 12 h light photoperiod for 7-10 days. Eighty-four tentative Verticillium sp. isolates were recovered and subcultured on modified water agar (WA-p) and PDA for identification (Inderbitzin et al. 2011, 2013). The majority of isolates (77 %) were identified as V. dahliae based on morphology; production of brown-pigmented microsclerotia and conidia features and dimensions (5.7 ± 0.9 μm long). Sequencing of mycelial DNA using primer pair ITS1-F and ITS4, resulted in consensus sequences of 503 bp. BLASTn analysis of ITS sequence of native isolate VdGi688 gave 100% identity to the ITS sequences of V. dahliae type strain PD322 (92% coverage) and Vd16_9 (100% coverage). In addition, 23% isolates morphologically corresponded to V. albo-atrum or V. nonalfalfae; melanized resting mycelia and round to oval-shaped conidia (5.2 ± 0.9 μm × 2.2 ± 0.5 μm). The ITS consensus sequence (544 bp) of native isolate VaaGi02 gave 99% identity (90-100 % coverage) to V. albo-atrum isolates CBS 127169, PSU 140, Vaa_TN1 and to V. nonalfalfae type PD592, CBS5451.88 and Vert 18. Sequences from isolates VdGi688 and VaaGi02 were deposited in GenBank as MW624723 and MW624724, respectively. Koch's postulates for seven V. dahliae isolates and eight V. albo-atrum isolates were fulfilled by injection of 1 mL of 1 x 107 conidia/mL suspension into the stem of A. altissima seedlings under greenhouse conditions. Six plants were inoculated per isolate in two independent experiments. Control plants were inoculated with sterile distilled water. All isolates caused leaf chlorosis, defoliation, and apical stem death, as well as internal necrosis and vascular discoloration. Control plants remained asymptomatic. The pathogens were re-isolated from internal symptomatic tissues of inoculated plants. To our knowledge, this is the first report of V. dahliae and V. albo-atrum sensu lato causing Verticillium wilt on A. altissima in Spain. The study suggests the potential of native isolates of Verticillum spp. in the biological control of ailanthus in the Mediterranean Basin. This work was funded by the Diputació de Girona (Spain) (2017/8719, 2019/3091, 2020/7565, and 2021/1468).

In vitro antimicrobial activity of methanolic extracts from cutaneous secretions of Amazonian amphibians against phytopathogens of agricultural interest

ABSTRACT The biochemical defense mechanisms of amphibians involve cutaneous secretions of bioactive molecules with antimicrobial activity. This study evaluated the in vitro activity of methanolic extracts from cutaneous secretions of two amphibian species of the Bufonidae family, Rhaebo guttatus and Rhinella marina, in the control of the phytopathogens Fusarium udum, Fusarium solani, Colletotrichum truncatum, Aspergillus flavus, Rhizoctonia solani, Macrophomina phaseolina, and Calonectria pseudometrosideri. The R. guttatus extract decreased the mycelial growth of F. udum, F. solani, A. flavus, and M. phaseolina at some tested concentrations. The R. marina extract decreased the mycelial growth of C. truncatum at the concentration of 0.5 mg mL-¹, and inhibited the mycelial growth of A. flavus at concentrations of 0.1 and 0.5 mg mL-¹, which was similar to the inhibition by the positive control. The R. marina extract also decreased the microsclerotia production by R. solani at concentrations of 0.2 and 0.3 mg mL-¹. In addition, the extracts inhibited conidial sporulation and germination at varying degrees. The inhibition of appressoria formation in C. truncatum by the R. guttatus and R. marina extracts was 85-99% and 63-100%, respectively. Our results demonstrated that treatment with extracts from R. guttatus and R. marina cutaneous secretions showed antifungal activity against the studied phytopathogens.

Physical, Cultural, and Chemical Alternatives for Integrated Management of Charcoal Rot of Strawberry.

Macrophomina phaseolina, the causal agent of charcoal rot in strawberry, induces plant wilting and collapse. The pathogen survives through the production of microsclerotia in the soil and in strawberry debris. However, its management is difficult, and the disease has become an increasing problem for the strawberry industry. Physical, cultural, and chemical alternatives for integrated management of charcoal rot were evaluated in laboratory and field trials during the 2017-18 and 2018-19 strawberry seasons. In a laboratory trial, M. phaseolina microsclerotia were subjected to heat treatment and germination was inhibited at 52, 56, 80, and 95°C after 30, 10, 1, and 0.5 min of exposure, respectively. In infected strawberry crowns, microsclerotial viability was reduced after 5 min, regardless of temperature, whereas in the field, reduction was observed after 1 min. In field trials, charcoal rot incidence of inoculated strawberry plants transplanted into white-striped plastic-mulched beds was reduced to 20.8%, compared with 60.8% for plants grown in the black plastic mulch. On commercial farms, crop residue removal from infested areas reduced the M. phaseolina population in the soil but did not decrease charcoal rot incidence. Moreover, M. phaseolina propagule densities in the soil and in strawberry debris was reduced by fumigant application at crop termination but surviving propagules allowed the population to increase over the summer. Furthermore, preplant fumigation with metam potassium reduced soil population and charcoal rot incidence. Overall, the adoption of integrated approaches such as physical, chemical, and/or cultural methods played a significant role in reducing M. phaseolina inoculum and contributed to control of the disease in areas with high disease pressure.

Production of Microsclerotia From Entomopathogenic Fungi and Use in Maize Seed Coating as Delivery for Biocontrol Against Fusarium graminearum

The commercial use of the entomopathogenic fungiMetarhiziumspp. in biopesticides has gained more interest since the discovery that several species of this genus are able to colonize roots. In general, commercial products withMetarhiziumare formulated based on conidia for insect pest control. The process of mass production, harvesting, and formulation of infective conidia can be detrimental for conidial viability. Entomopathogenic fungi such asMetarhiziumspp. are able to produce high concentrations of resistant structures, known as microsclerotia, when grown in liquid media. Microsclerotia are desiccation tolerant, with excellent storage stability, and are capable of producing high quantities of infective conidia after rehydration. The aim of this study was to evaluate microsclerotia production by different isolates ofMetarhiziumspp. and determine the effect of microsclerotia coated onto maize seeds on plant growth in the presence of soil-borne pathogenFusarium graminearum. On average, ~1 × 105microsclerotia/mL were produced by selected isolates ofM. anisopliae(A1080 and F672) andMetarhizium robertsii(F447). Microsclerotia were formulated as granules with diatomaceous earth and used for seed coating, after which propagules produced around 5 × 106CFU/g of seeds. In the presence of the plant pathogen, maize plants grown from untreated seeds had the lowest growth, while plants treated with theMetarhiziummicrosclerotia had significantly greater growth than the control plants. Hyphae were observed growing on and in root tissues in all theMetarhiziumspp. treatments but not in samples from control plants.Metarhiziumhyphal penetration points' on roots were observed 1 month after sowing, indicating the fungi were colonizing roots as endophytes. The results obtained indicate that microsclerotia can be coated onto seeds, providing plant protection against soil plant pathogens and a method to establishMetarhiziumin the ecto- and endo-rhizosphere of maize roots, allowing the persistence of this biocontrol agent.

Potential of few fungicides and plant extracts for managing charcoal rot of soybean caused by Macrophomina phaseolina (Tassi) Gold. in Madhya Pradesh, India

Charcoal rot disease of soybean caused by Macrophomina phaseolina is a serious problem in most of the soybean growing area of Madhya Pradesh. In this study, seven plant extracts viz., leaves of Azadirachta indica, Citrus limon, Polyalthia longifolia, Parthenium hysterophorus and Ricinus communis, bulb of Allium sativum and Allium cepa and eight fungicides viz., Captan (0.25%), Mancozeb (0.25%), Carbendazim + Mancozeb (25%), Thiophanate Methyl (0.1%), Pyraclostrobin (0.2%), Carbendazim (0.1%) and Blue copper (0.3% ) were evaluated for an effective management of charcoal rot of soybean caused by M. phaseolina (Tassi) Goid under in vitro and in vivo condition. Among plant extracts, garlic clove extract was found most effective showing 77.3 % growth inhibition and poor microslerotia formation of M. phaseolina by 77.3 % followed by parthenium leaf extract (75.2% inhibition) at 15 % concentration. Two soil drenching of garlic clove extracts @ 15.0 % concentration also found most effective for the management of disease under field condition recorded minimum disease incidence (13.5%) and highest yield (14.6q/ha). Among fungicides, Carbendazim (0.1%) and Thiophanate Methyl (0.1%) showed 100 % inhibition of radial growth and microsclerotia production of M phaseolina under in vitro condition. Two soil drenching of Carbendazim @ 0.1% found to be most effective for the management of charcoal rot of soybean under field condition showing minimum disease incidence (5.36%) and producing highest yield (16.0 q/ha) followed by Thiophanate Methyl. These results suggested that the toxic effect of Carbendazim and Thiophanate Methyl and A. sativum inhibited maximum mycelium growth in vitro and provide management of charcoal rot disease under field conditions.

Molecular Mechanisms Controlling the Disease Cycle in the Vascular Pathogen Verticillium dahliae Characterized Through Forward Genetics and Transcriptomics.

The soil-borne pathogen Verticillium dahliae has a worldwide distribution and a plethora of hosts of agronomic value. Molecular analysis of virulence processes can identify targets for disease control. In this work, we compared the global gene transcription profile of random T-DNA insertion mutant strain D-10-8F, which exhibits reduced virulence and alterations in microsclerotium formation and polar growth, with that of the wild-type strain. Three genes identified as differentially expressed were selected for functional characterization. To produce deletion mutants, we developed an updated version of one-step construction of Agrobacterium-recombination-ready plasmids (OSCAR) that included the negative selection marker HSVtk (herpes simplex virus thymidine kinase gene) to prevent ectopic integration of the deletion constructs. Deletion of VdRGS1 (VDAG_00683), encoding a regulator of G protein signaling (RGS) protein and highly upregulated in the wild type versus D-10-8F, resulted in phenotypic alterations in development and virulence that were indistinguishable from those of the random T-DNA insertion mutant. In contrast, deletion of the other two genes selected, vrg1 (VDAG_07039) and vvs1 (VDAG_01858), showed that they do not play major roles in morphogenesis or virulence in V. dahliae. Taken together the results presented here on the transcriptomic analysis and phenotypic characterization of D-10-8F and ∆VdRGS1 strains provide evidence that variations in G protein signaling control the progression of the disease cycle in V. dahliae. We propose that G protein-mediated signals induce the expression of multiple virulence factors during biotrophic growth, whereas massive production of microsclerotia at late stages of infection requires repression of G protein signaling via upregulation of VdRGS1 activity.

Microsclerotia production of Metarhizium spp. for dual role as plant biostimulant and control of Spodoptera frugiperda through corn seed coating

The fungal genus Metarhizium comprises entomopathogenic species capable of producing overwintering structures known as microsclerotia. These structures offer many advantages in pest control due to the formation of infective conidia in situ and their persistence in the environment under adverse conditions. In addition, the in vitro production of Metarhizium microsclerotia under controlled liquid fermentation is faster and with greater process control than the production of aerial conidia. However, the potential of Metarhizium microsclerotia to control pests from the orders Lepidoptera and Hemiptera is unexplored. In this study, we examined the ability of Metarhizium spp. microsclerotia to promote corn growth and to provide plant protection against Spodoptera frugiperda (Lepidoptera: Noctuidae) and Dalbulus maidis (Hemiptera: Cicadellidae), through seed coating using microsclerotial granules. A screening to find higher microsclerotia producers was conducted by culturing 48 native Brazilian isolates of Metarhizium spp. (Metarhizium anisopliae, Metarhizium robertsii, Metarhizium humberi and Metarhizium sp. indeterminate). The best microsclerotia producers, M. anisopliae ESALQ1814, M. robertsii ESALQ2450 and M. humberi ESALQ1638 improved the leaf area, plant height, root length, and dry weight of plants compared to un-inoculated plants. Significant reduction in S. frugiperda survival (mortality > 55% after 7 days) was observed when larvae were fed on corn plants treated with any of the three Metarhizium species. Conversely, survival of D. maidis adults were unaffected by feeding on fungus-inoculated plants. Our results suggest that microsclerotia of Metarhizium spp. may act as biostimulants and to provide protection against S. frugiperda in corn through seed coating, thus adding an innovative strategy into the integrated management of this major worldwide pest.

The Vta1 transcriptional regulator is required for microsclerotia melanization in Verticillium dahliae

Many fungi are able to produce resting structures, which ensure survival and protect them against various stresses in their habitat such as exposure to UV light, temperature variations, drought as well as changing pH and nutrient conditions. Verticillium dahliae is a plant pathogenic fungus that forms melanized resting structures, called microsclerotia, for survival of time periods without a host. These highly stress resistant microsclerotia persist in the soil for many years and are therefore problematic for an effective treatment of the fungus. The Verticillium transcription activator of adhesion 1 (Vta1) was initially identified as one of several transcriptional regulators that rescue adhesion in non-adhesive Saccharomyces cerevisiae cells. Vta2 and Vta3 are required for early steps in plant infection and colonization and additionally control microsclerotia formation. Here, we show that Vta1 function is different, because it is dispensable for root colonization and infection. Vta1 is produced in the fungal cell during microsclerotia development. Analysis of the deletion mutant revealed that the absence of Vta1 allows microsclerotia production, but they are colorless and no more melanized. Vta1 is required for melanin production and activates transcription of melanin biosynthesis genes including the polyketide synthase encoding PKS1 and the laccase LAC1. The primary function of Vta1 in melanin production is important for survival of microsclerotia as resting structures of V. dahliae.

Phylogenetic Diversity and Effect of Temperature on Pathogenicity of Colletotrichum lupini.

Although lupin anthracnose caused by Colletotrichum lupini is a significant threat for spring and winter lupin crops, it has been poorly studied so far. This study aimed at characterizing the (i) phylogenetic, (ii) morphological, and (iii) physiological diversity of collected isolates from anthracnose-affected lupins. The genetic identification of representative isolates (n = 71) revealed that they were all C. lupini species, further confirming that lupin anthracnose is caused by this species. However, multilocus sequencing on these isolates and 16 additional reference strains of C. lupini revealed a separation into two distinct genetic groups, both of them characterized by a very low genetic diversity. The diversity of morphological characteristics of a selected subset of C. lupini isolates was further evaluated. To the best of our knowledge, microsclerotia production observed for some isolates has never been reported so far within the Colletotrichum acutatum species complex. Finally, the modeling of growth responses of a subset of C. lupini strains revealed the capacity of some strains to grow in vitro at 5°C. This ability was also evidenced in planta, because C. lupini DNA was detectable in plants from 14 days postinoculation at 5°C onward, whereas symptoms began to appear a week later, although at a very low level. Since lupin crops are planted during winter or early spring, growth studies in vitro and in planta demonstrated the capability of the species to grow at temperatures ranging from 5 to 30°C, with an optimum close to 25°C. In this study, C. lupini-specific primers were also designed for real-time quantitative PCR on fungal DNA and allowed the detection of C. lupini in asymptomatic field samples. These results open perspectives to detect earlier and limit the development of this pathogen in lupin crops.

Sources of Inoculum and Survival of Macrophomina phaseolina in Florida Strawberry Fields.

Macrophomina phaseolina, the causal agent of charcoal rot, affects strawberry crowns, inducing plant collapse. The fungus survives in the soil through the production of microsclerotia and is usually controlled by preplant fumigation of soil. However, in the 2016 to 2017 Florida strawberry season, even after soil fumigation, about 30% plant mortality still occurred in plastic-covered beds that were used for a second season and where crop residue (mainly old strawberry crowns) was disposed of between beds. Therefore, this study was conducted to determine if M. phaseolina can survive on strawberry debris over summer in Florida and if so, verify whether strawberry debris might act as a source of inoculum for new transplants. Crowns from the previous season were collected from commercial farms where charcoal rot had been reported, and M. phaseolina was recovered from all samples. In a research field, infected crowns were buried in the soil at different depths and retrieved every 2 weeks during the summer. After 8 weeks, M. phaseolina could be recovered at all depths. Moreover, inoculation of strawberry plants by drenching the soil, dipping roots, or spraying leaves with a M. phaseolina microsclerotial suspension from pure cultures or infected crowns produced symptoms with differences in incubation periods depending on cultivar susceptibility. Furthermore, infected crowns disposed of in the aisles between beds or buried next to new transplants of cultivars Strawberry Festival, Florida Beauty, and Winterstar induced charcoal rot, with the level of aggressiveness depending on the cultivar susceptibility and inoculum placement in the field.

Insights into VdCmr1‐mediated protection against high temperature stress and UV irradiation in Verticillium dahliae

The fungus Verticillium dahliae causes vascular wilt disease on more than 200 plant species worldwide. This fungus can survive for years in soil as melanized microsclerotia. We found that VdCmr1, a transcription factor, is required for the melanin production and increased survival following UV irradiation in V. dahliae but not for microsclerotia production or virulence. Here, we provided evidence how VdCmr1 protects against high temperature (HT) and UV irradiation in V. dahliae. The results indicate that VdCmr1 mediates entry to the diapause period in V. dahliae in response to HT and contributes to the expression of proteins to minimize protein misfolding and denaturation. VdCmr1 deletion results in the misregulation of DNA repair machinery, suggestive of reduced DNA repair capacity following UV irradiation and in correlation with the low survival rate of UV-treated VdCmr1 mutants. We discovered a putative VdCmr1-dependent gene cluster associated with secondary metabolism and stress responses. We also functionally characterized two VdCmr1-responsive genes participating in HT and UV response. These results shed further light on the roles of VdCmr1 in protection from HT or UV irradiation, and the additional insights into the mechanisms of this protection may be useful to exploit for more effective disease control.