Germ Tube Growth Research Articles

Characterization of Bipolaris bicolor germination: effects of a physical factor on fungal adaptability.

Studies on physiological responses to stimuli from physical factors are essential for understanding the dynamics of the microorganisms and higly important for the management of plant diseases. Besides, the development of an epidemiological model for pathogen populations requires studying their physiological responses to physical stimuli. The objective of this study was to evaluate the germination dynamics of spores from six isolates of Bipolaris bicolor under effects of light at 25°C. Suspensions of 1.6 × 105 conidia mL- 1 from the B. bicolor isolates were inoculated onto Petri dishes containing agar-water culture medium and incubated in a BOD chamber under two physical conditions: (a) constant darkness and (b) constant light for five hours. The study was conducted in a completely randomized design, with a 6 × 2 factorial arrangement (six B. bicolor isolates and two physical conditions) and five replications. The length of the germ tube was measured hourly. The constant darkness resulted in higher mean germ tube growth for the pathogen; however, differences in the final germination percentage were found among the isolates. The isolate F-24-02 exhibited the highest germination adaptability to constant darkness, presenting the longest germ tube length.

The systemic herbicide glyphosate affects the sporulation dynamics of Rhizophagus species more severely than mechanical defoliation or the contact herbicide diquat.

Arbuscular mycorrhizal fungi (AMF) are totally dependent on a suitable host plant for their carbon resources. Here, we investigated under in vitro conditions, the impact of defoliation practices, i.e., mechanical defoliation or chemical defoliation with a contact herbicide (Reglone®, containing the active ingredient diquat) or systemic herbicide (RoundUp®, containing the active ingredient glyphosate),on the dynamics of spore production of Rhizophagus irregularis and Rhizophagus intraradices associated with Solanum tuberosum and/or Medicago truncatula. Glyphosate affected the spore production rate more rapidly and severely than diquat or mechanical defoliation. We hypothesize that this effect was related to disruption of the C metabolism in the whole plant combined with a possible direct effect of glyphosate on the fungus within the roots and/or perhaps in soil via the release of this active ingredient from decaying roots. No glyphosate could be detected in the roots due to technical constraints, while its release from the roots in the medium corresponded to 0.11% of the active ingredient applied to the leaves. The three defoliation practices strongly affected root colonization, compared to the non-defoliated plants. However, the amount of glyphosate released into the medium did not affect spore germination and germ tube growth. These results suggest that the effects of defoliation on the dynamics of spore production are mainly indirect via an impact on the plant, and that the effect is faster and more marked with the glyphosate-formulation, possibly via a direct effect on the fungus in the roots and more unlikely on spore germination.

Characterization and fungicide sensitivity of Gnomoniopsis fructicola causing Gnomonia Leaf Blotch of strawberry in the Carolinas.

Emerging fungal pathogens have always been an issue of concern in southeastern U.S. strawberry production. In 2023, an unusual outbreak of Gnomonia leaf blotch occurred at one North Carolina (NC) and multiple South Carolina (SC) strawberry farms and marked the first report of its occurrence in SC. Molecular identification and phylogenetic analysis of isolates from multiple locations identified the fungus Gnomoniopsis fructicola as the causal agent. In vitro germination of G. fructicola progressed slowly and remained less than 40% even after 24 h of incubation. Similarly, germ tube growth was slow compared to other pathogens. Slow symptom development on strawberry leaves of young strawberry plants grown in the greenhouse started 5 weeks after inoculation. Once the pathogen established on greenhouse plants, leaf necrosis forming blotches was observed. The baseline sensitivity of G. fructicola isolates to commonly used chemical classes of fungicides was assessed. Propiconazole, cyprodinil, pyraclostrobin, and fludioxonil were highly effective in mycelial growth assays with EC50 values < 0.01 µg/ml. Iprodione and thiophanate-methyl were also effective with EC50 values ranging from 0.05 to 1.38 and 2.01 to 23.96 µg/ml, respectively. Fluopyram and fenhexamid were ineffective with EC50 values >100 µg/ml. Based on conversations with the producers, the disease outbreak was linked to transplants from the same nursery source. This study reports for the first time the presence of Gnomonia leaf blotch in South Carolina and provides valuable insights into chemical management options for G. fructicola.

The rice endophytic bacterium Bacillus velezensis LS123N provides protection against multiple pathogens and enhances rice resistance to wind with increase in yield

Rice (Oryza sativa) is a globally important staple food, but its production was influenced by various biotic and abiotic stresses. This study involved screening endophytic bacterial strains for their antagonistic activities and control efficacy against multiple rice pathogens in planta assays. Subsequent greenhouse investigations identified Bacillus velezensis LS123N as the most promising strain for effectively controlling various rice diseases, including bacterial blight, rice blast, bakanae disease, Pythium disease, and brown spot disease. Field trials further demonstrated that LS123N significantly reduced naturally occurring brown spot disease caused by Bipolaris oryzae in both seedlings and mature rice plants, and increased rice yields. For disease control against bacterial blight, Pythium disease, and brown spot, only a single treatment during seed germination resulted in significant disease control efficacy. The application of LS123N during seed germination aligns with common rice cultivation practices. Additionally, LS123N exhibited the capacity to improve rice resistance to wind stress and enhance rice growth during the seedling stage. Endophytic colonization of LS123N was evidenced from seed to mature rice plants after seed treatment. Interestingly, the expression of many rice defense genes was suppressed within 48 h of the LS123N introduction. In vitro assays showed that LS123N inhibited spore germination and normal growth of germ tubes of B. oryzae, produced multiple hydrolases, siderophores, and IAA, and had the ability to solubilize phosphorus compounds. In summary, these results indicate that B. velezensis LS123N is a promising biocontrol agent and has the potential to be developed into a biofungicide, biofertilizer, and biostimulator.

Carboxymethyl cellulose/Tween 80/Litsea cubeba essential oil nanoemulsion inhibits the growth of Penicillium digitatum and extends the shelf-life of ‘Shatangju’ mandarin

Penicillium digitatum (P. digitatum) is the primary pathogenic fungus responsible for postharvest citrus green mold, leading to a deterioration in fruit quality and significant economic losses. Litsea cubeba essential oil (LCEO) has gained widespread attention as a natural antifungal substance due to its high safety profile, non-resistance traits, and broad-spectrum antifungal activity. In this study, we prepared Litsea cubeba essential oil nanoemulsion (LCEO-NE) using Tween 80 and carboxymethyl cellulose (CMC) as emulsifiers through ultrasonic emulsification. The investigation delved into their stability, rheology, inhibitory activity against P. digitatum, and efficacy in preserving ‘Shatangju’ mandarin during storage. The results showed that the droplet size, polydispersity index, and zeta potential were 18.69 nm, 0.116, and −29.72 mV, respectively. LCEO-NE added with CMC and Tween 80 synergistically (LCEO-NE-CMC) have exhibited higher pH stability, ionic strength stability, thermal stability, storage stability, and centrifugal stability compared to LCEO-NE with Tween 80. In vitro fungistatic tests revealed that the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of LCEO-NE-CMC against P. digitatum were 1 μL mL−1 and 2 μL mL−1, respectively. LCEO-NE-CMC effectively inhibited mycelium growth, spore germination, and elongation of germ tubes. Scanning electron microscopy results demonstrated that LCEO-NE-CMC caused wrinkling and twisting of P. digitatum mycelium. Transmission electron microscopy results further revealed severe damage to the cell membrane, cytoplasm, and organelles, hindering mycelial growth. Furthermore, LCEO-NE-CMC treatment of ‘Shatangju’ mandarin significantly reduced weight loss and decay rate and delayed the decline in hardness and total soluble solids. In summary, LCEO-NE-CMC has been validated as a potent antifungal agent capable of extending the shelf life of ‘Shatangju’ mandarin.

Study on the inhibitory effect of fermentation extract of Microporus vernicipes on Candida albicans.

Candida albicans is one of the most common species of Candida, which cause various mucosal and systemic infectious diseases. However, the resistance rate to existing clinical antifungal drugs gradually increases in C. albicans. Therefore, new antifungal drugs must be developed to solve the current problem. This study discovered that the solid fermented ethyl acetate crude extract of Microporus vernicipes had inhibitory activity on C. albicans. This study determined that the Mv5 components had significantly inhibited the activity of C. albicansusing column chromatography separation component screening. The components included 23 compounds of fatty acids and their derivatives, alkaloids, phenols, and other classes using ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HR-MS) analysis, with fatty acids constituting the primary components. The mechanism of action showed that the minimum inhibitory concentration (MIC) of Mv5 components against C. albicans was 15.63μg/mL, while minimum fungicidal concentration (MFC) was 31.25μg/mL. Mv5 components can inhibit the early biofilm formation and destroy the mature biofilm structure. It can inhibit the germ tube growth of C. albicans, thereby inhibiting the transformation of yeast morphology to hyphae. We detected 193 differentially expressed genes, including 156 upregulated and 37 downregulated genes in the Mv5 components of the MIC concentration group. We detected 391 differentially expressed genes, including 334 upregulated and 57 downregulated expression genes in the MFC concentration group. Among these differentially expressed genes, the genes related to mycelium and biofilm formation were significantly downregulated. GO enrichment analysis presented that single-organism process metabolic process, and cellular processes were the biological processes with the most gene enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG)of Mv5 components were mainly enriched in metabolic pathways, such as meiosis yeast and amino acid metabolism. Therefore, it is believed that the fermentation extract of M. vernicipes inhibits C. albicans, which can provide clues for developing effective antifungal drugs.

Novel Endophytic Pseudescherichia sp. GSE25 Strain Significantly Controls Fusarium graminearum and Reduces Deoxynivalenol in Wheat.

Fusarium heading blight (FHB) is a devastating disease in wheat, primarily caused by field invasion of Fusarium graminearum. Due to the scarcity of resistant wheat varieties, the agricultural sector resorts to chemical fungicides to control FHB incidence. On the other hand, biocontrol represents a promising, eco-friendly approach aligned with sustainable and green agriculture concepts. In the present study, a bacterial endophyte, Pseudescherichia sp. (GSE25), was isolated from wheat seeds and identified through complete genome sequencing and phylogenetic analysis. In vitro testing of this endophytic strain demonstrated strong antifungal activity against F. graminearum PH-1 by inhibiting spore germination, suppressing germ tube growth, and causing cell membrane damage. Under field conditions, the strain GSE25 significantly reduced the FHB incidence and the associated deoxynivalenol mycotoxin accumulation by over 60% and 80%, respectively. These findings highlight the potential of the isolated bacterial endophyte Pseudescherichia sp. GSE25 strain as a biocontrol agent in protecting wheat from FHB-caused F. graminearum. This is the first report showing a biocontrol effect of Pseudescherichia sp. a strain against phytopathogens.

Synthesis and evaluation of imidazo[1,2-a]quinoxaline derivatives as potential antifungal agents against phytopathogenic fungi.

To discover novel and effective potential agricultural antifungal agents, various kinds of imidazo[1,2-a]quinoxaline derivatives were designed, and synthesized from available and inexpensive reagents. Their antifungal activities were first evaluated against ten typical phytopathogenic fungi. The in vitro antifungal activity showed that some compounds exhibited more obvious broad-spectrum fungicidal activity than the two commercially-available fungicides chlorothalonil and hymexazol. Valsa mali and Botrytis cinerea strains exhibited the highest susceptibility with EC50 values of 1.4-27.0μg/mL to more than ten compounds. Compounds 5c and 5f showed the most promising inhibitory effects against Valsa mali (EC50 = 5.6μg/mL) and Fusarium solani (EC50 = 5.1μg/mL), respectively. Preliminary studies on the mechanism of action indicated that the imidazo[1,2-a]quinoxaline skeleton likely exerted its antifungal effects by disrupting hyphal differentiation, spore germination, and germ tube growth. Moreover, the cell experiment results indicated that these target compounds possessed good safety to BV2 cells. Overall, compounds 5c and 5f can be considered candidate compounds against specific fungi for further detailed research. This study can provide a theoretical basis for the application of imidazo[1,2-a]quinoxaline scaffolds as novel fungicides in agriculture.

Fungicide Sensitivity in the European Canker Fungus, Neonectria ditissima

Neonectria ditissima is one of the most serious pathogens of apple trees in temperate climatic zones, causing bark canker as well as pre- and post-harvest fruit rots. Control of fruit rots in particular is hampered by uncertainty about the sensitivity to, and efficacy of, available fungicides. Using 43 N. ditissima isolates from Germany and South Tyrol, the effective concentrations causing a 50% inhibition of germ-tube growth (EC50) were determined for fludioxonil (0.009–0.046 ppm), trifloxystrobin (0.018–0.43 ppm), thiophanate-methyl (2.05–15.9 ppm), dodine (0.61–6.33 ppm), cyprodinil (5.04–83.7 ppm), pyrimethanil (22.0–500 ppm), fluopyram (1.35–140 ppm) and boscalid (> 500 ppm). In a detached-fruit assay based on artificial wound inoculation of apples previously dipped in fungicides at commercial dosages, fludioxonil and, to a lesser extent, fluopyram and dodine significantly reduced the fruit rot lesion size, whereas trifloxystrobin, cyprodinil and boscalid did not. Lesion size was also directly correlated to the amount of conidial inoculum, whereby wounds inoculated with 5000 conidia gave rise to larger lesions than those with 500 or 50 conidia. In a field trial of apple trees artificially inoculated with N. ditissima conidia at flowering, fludioxonil gave good control of blossom-end rot, and trifloxystrobin gave limited control, whereas cyprodinil was ineffective. Therefore, the efficacies of different fungicides were broadly in line with the EC50 values determined under laboratory conditions.

Herbicide 2,4-dichlorophenoxyacetic acid interferes with MAP kinase signaling in Fusarium graminearum and is inhibitory to fungal growth and pathogenesis

Plant hormones are important for regulating growth, development, and plant-pathogen interactions. Some of them are inhibitory to growth of fungal pathogens but the underlying mechanism is not clear. In this study, we found that hyphal growth of Fusarium graminearum was significantly reduced by high concentrations of IAA and its metabolically stable analogue 2,4-dichlorophenoxyacetic acid (2,4-D). Besides inhibitory effects on growth rate, treatments with 2,4-D also caused significant reduction in conidiation, conidium germination, and germ tube growth. Treatments with 2,4-D had no obvious effect on sexual reproduction but significantly reduced TRI gene expression, toxisome formation, and DON production. More importantly, treatments with 2,4-D were inhibitory to infection structure formation and pathogenesis at concentrations higher than 100 µM. The presence of 1000 µM 2,4-D almost completely inhibited plant infection and invasive growth. In F. graminearum, 2,4-D induced ROS accumulation and FgHog1 activation but reduced the phosphorylation level of Gpmk1 MAP kinase. Metabolomics analysis showed that the accumulation of a number of metabolites such as glycerol and arabitol was increased by 2,4-D treatment in the wild type but not in the Fghog1 mutant. Transformants expressing the dominant active FgPBS2S451D T455D allele were less sensitive to 2,4-D and had elevated levels of intracellular glycerol and arabitol induced by 2,4-D in PH-1. Taken together, our results showed that treatments with 2,4-D interfere with two important MAP kinase pathways and are inhibitory to hyphal growth, DON biosynthesis, and plant infection in F. graminearum.

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa

In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.

Germination and Invasion of Paraphoma radicina on Roots of a Susceptible and a Resistant Alfalfa Cultivar (Medicago sativa).

Alfalfa Paraphoma root rot (APRR) (Paraphoma radicina) is a recently described alfalfa disease widely distributed in China, first reported in 2020. So far, the resistance levels of 30 alfalfa cultivars to APRR have been characterized; however, the resistance mechanisms among these cultivars remain unknown. In the present study, the alfalfa resistance mechanisms against APRR were investigated by studying the difference of P. radicina infecting susceptible (Gibraltar) and resistant (Magnum II) alfalfa cultivars under the light microscope and scanning electronic microscope. The conidial germination and germ tube growth in the root exudates of different resistant cultivars were also compared. The results revealed that conidial germination, germ tube development, and P. radicina penetration into root tissues of resistant plants were delayed. In susceptible and resistant cultivars, P. radicina infected roots by penetrating epidermal cells and the intercellular space between epidermal cells. During the infection process, germ tubes penetrated the root surface directly or formed appressoria. However, the penetration percentage on the susceptible cultivar was significantly higher than on the resistant cultivar, irrespective of the infection route. Moreover, disintegrated conidia and germ tubes were observed on resistant cultivar roots at 48 h postinoculation. The conidial germination and germ tube growth in root exudates of susceptible cultivars were significantly higher than in resistant cultivars. The current findings implied that the alfalfa resistance mechanism might be related to root exudates. These findings could provide insights into the alfalfa resistance mechanism following P. radicina infection.

The C2H2 transcription factor SltA is required for germination and hyphal development in Aspergillus fumigatus.

Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination.IMPORTANCEAspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

Effect of temperature on sporulation and spore development of giant kelp (Macrocystis pyrifera).

Rising ocean temperature is a major driver of kelp forest decline worldwide and one that threatens to intensify over the coming decades. What is not particularly well understood are the mechanisms that drive loss and how they operate at differing life stages. This study aimed to establish an understanding of the effects of increasing temperature on the early developmental stages of the giant kelp, Macrocystis pyrifera. Sporulation was carried out across 10 temperature treatments from 9.5 to 26.2°C ± 0.2°C at approximately 2°C intervals. Spores were then incubated at these temperatures under a 20.3±1.7 μmol photons m-2 s-1, 16L:8D photoperiod for 5 days. Results indicate that spore release was positively correlated with increasing temperature, whereas an inverse trend was observed between temperature and the growth of germ-tube. The thermal threshold for spore and germling development was determined to be between 21.7°C and 23.8°C. Spore settlement was the most drastically effected developmental phase by increasing temperature. This study highlights the vulnerability of early life stages of M. pyrifera development to rising ocean temperature and has implications for modelling future distribution of this valuable ecosystem engineer in a changing ocean.

Biocontrol of strawberry gray mold caused by Botrytis cinerea with the termite associated Streptomyces sp. sdu1201 and actinomycin D.

Strawberry gray mold caused by Botrytis cinerea is one of the most severe diseases in pre- and post-harvest periods. Although fungicides have been an effective way to control this disease, they can cause serious "3R" problems (Resistance, Resurgence and Residue). In this study, Streptomyces sp. sdu1201 isolated from the hindgut of the fungus-growing termite Odontotermes formosanus revealed significant antifungal activity against B. cinerea. Four compounds (1-4) were isolated from Streptomyces sp. sdu1201 and further identified as actinomycins by the HRMS and 1D NMR data. Among them, actinomycin D had the strongest inhibitory activity against B. cinerea with the EC50 value of 7.65 μg mL-1. The control effect of actinomycin D on strawberry gray mold was also tested on fruits and leaves in vitro, and its control efficiency on leaves was 78.77% at 3 d. Moreover, actinomycin D can also inhibit the polarized growth of germ tubes of B. cinerea. Therefore, Streptomyces sp. sdu1201 and actinomycin D have great potential to gray mold as biocontrol agents.

Elucidating the effect of tomato leaf surface microstructure on Botrytis cinerea using synthetic systems.

For some pathogenic fungi, sensing surface topography is part of their infection strategy. Their directional growth and transformation to a new developmental stage is influenced by contact with topographic features, which is referred to as thigmo-response, the exact functionality of which is not fully understood. Research on thigmo-responses is often performed on biomimetically patterned surfaces (BPS). Polydimethylsiloxane (PDMS) is especially suitable for fabrication of BPS. Here, we used synthetic BPS surfaces, mimicking tomato leaf surface, made from PDMS with the pathogenic fungus Botrytis cinerea to study the influence of structural features of the leaf surface on the fungus behavior. As a control, a PDMS surface without microstructure was fabricated to maintain the same chemical properties. Pre-penetration processes of B. cinerea, including the distribution of conidia on the surface, germination, and germ tube growth were observed on both leaf-patterned and flat PDMS. Microstructure affected the location of immediate attachment of conidia. Additionally, the microstructure of the plant host stimulated the development of germ tube in B. cinerea, at a higher rate than that observed on flat surface, suggesting that microstructure plays a role in fungus attachment and development.

The Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana for Management of the Melon Fly Zeugodacus cucurbitae: Pathogenicity, Horizontal Transmission, and Compatability with Cuelure

Simple SummaryThe melon fly Zeugodacus cucurbitae is an invasive fruit fly that causes extensive damage to many fruit crops. Sustainable management of this pest can be achieved through environmentally friendly and safely integrated pest management (IPM), in which the use of biopesticides is a key component. In this study, various isolates of entomopathogenic fungi were evaluated on two developmental stages of this pest. The fungal isolate ICIPE 69 has the greatest potential. The study also evaluated whether ICIPE 69 could be used together with the male pheromone of the melon fruit fly, as well as whether infected flies could contaminate clean flies. Our study indicates that ICIPE 69 can be included in IPM packages to reduce yield losses.In the laboratory, the pathogenicity of thirteen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin against the melon fly Zeugodacus cucurbitae (Coquillett) were assessed by exposing adults to 0.3 g of dry conidia (~3 × 109 conidia) of each isolate for 5 min and monitoring mortality for up to 5 days. Compatibility with a male pheromone, cuelure, (4-(p-acetoxyphenyl)-2-butanone), was determined by testing conidial germination and germ tube growth of the most promising isolate, M. anisopliae ICIPE 69, in the presence of cuelure at different temperatures. For horizontal transmission, the flies were separated by sex, separately exposed to M. anisopliae ICIPE 69, and subsequently mixed with non-exposed flies from the other sex. The most pathogenic isolates were M. anisopliae ICIPE 69, 18, and 30, causing mortalities of 94, 87, and 81%, with 5 days post-exposure, respectively. Metarhizium anisopliae ICIPE 69 caused the highest pupal mortality of 74%, with 15 days post-exposure. Horizontal transmission of M. anisopliae ICIPE 69 among male and female Z. cucurbitae was confirmed by 59 and 67% mortality after exposure to infected donor males and females, respectively. Metarhizium anisopliae ICIPE 69 affected the oviposition, but not hatchability, of infected Z. cucurbitae females. Metarhizium anisopliae ICIPE 69 is, therefore, a potential isolate for biopesticide development for Z. cucurbitae management in cucurbit production systems.

Antifungal Activity of Mexican Propolis on Clinical Isolates of Candida Species.

Infections caused by micro-organisms of the genus Candida are becoming a growing health problem worldwide. These fungi are opportunistic commensals that can produce infections—clinically known as candidiasis—in immunocompromised individuals. The indiscriminate use of different anti-fungal treatments has triggered the resistance of Candida species to currently used therapies. In this sense, propolis has been shown to have potent antimicrobial properties and thus can be used as an approach for the inhibition of Candida species. Therefore, this work aims to evaluate the anti-Candida effects of a propolis extract obtained from the north of Mexico on clinical isolates of Candida species. Candida species were specifically identified from oral lesions, and both the qualitative and quantitative anti-Candida effects of the Mexican propolis were evaluated, as well as its inhibitory effect on C. albicans isolate’s germ tube growth and chemical composition. Three Candida species were identified, and our results indicated that the inhibition halos of the propolis ranged from 7.6 to 21.43 mm, while that of the MFC and FC50 ranged from 0.312 to 1.25 and 0.014 to 0.244 mg/mL, respectively. Moreover, the propolis was found to inhibit germ tube formation (IC50 ranging from 0.030 to 1.291 mg/mL). Chemical composition analysis indicated the presence of flavonoids, including pinocembrin, baicalein, pinobanksin chalcone, rhamnetin, and biochanin A, in the Mexican propolis extract. In summary, our work shows that Mexican propolis presents significant anti-Candida effects related to its chemical composition, and also inhibits germ tube growth. Other Candida species virulence factors should be investigated in future research in order to determine the mechanisms associated with antifungal effects against them.

A novel antimicrobial peptide derived from human BPIFA1 protein protects against Candida albicans infection.

Bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1) is an innate immunity defense protein. Our previous studies proved its antibacterial and antiviral effects, but its role in fungi remains unknown. The study aimed to identify antifungal peptides (AFP) derived from BPIFA1, and three antimicrobial peptides (AMP1–3) were designed. The antifungal effects were proved by growth inhibition assay. AMP3 activity was confirmed by germ tube growth experiment and XTT assay. Its effects on cell wall and membrane of Candida albicans were assessed by tannic acid and Annexin V-FITC/PI double staining, respectively. Additionally, scanning electron microscope (SEM) and transmission electron microscopy (TEM) were used for morphological and ultrastructural observation. The expression of ALS1, EAP1, and SUN41 was tested by qPCR. Ultimately, three AMPs could fight against C. albicans in vitro, and AMP3 was highly effective. It functioned by destroying the integrity of cell wall and normal structure of cell membrane. It also inhibited biofilm formation of C. albicans. In addition, AMP3 down-regulated the expression of ALS1, EAP1, and SUN41, those are known to be involved in virulence of C. albicans. Altogether, the study reported successful development of a novel AFP, which could be used as a new strategy for antifungal therapy.

Thevetia peruviana (Pers.) K. Schum. Potential Antifungal Agent Against Mycosphaerella fijiensis Morelet, Fungi Responsible of Black Leaf Streak Disease (BLSD) of Plantain (Musa spp)

Alternatives to synthetic chemicals are undertaken against phytopathogens. The aim of this work is to evaluate the effect of seed extracts of Thevetia peruviana (Pers.) K. Schum. on Mycosphaerella fijiensis Morelet, fungus responsible for banana black leaf streak disease. Five extracts of T. peruviana, hexane extract (HE), ethyl acetate extract (EAE), acetone extract (AcE), methanol extract (ME) and aqueous extract (AqE), and a fungicide, Azoxystrobin were used. GC-MS of acetone extract was performed. Fifty (50) strains of M. fijiensis per sampling site were tested. Three concentrations of extracts 6.25 (C1), 12.5 (C2), and 25 (C3) μl/ml, a negative control (0 μl/ml) and 10 ppm of azoxystrobin were used for the tests. The MIC50 and MIC90 were determined. GC-MS showed chemical compounds with different molecular height such as acids, sugars, and esters. AcE and AqE significantly reduced M. fijiensis germ tube growth at C2 and C3 concentrations and with inhibition percentage respectively ranged of 60-90% and 40-80%. The growth levels of the germ tubes were above the strobilurin resistance threshold at Njombe and peasant plantation, ranging from 77.9% to 92.3%. AcE showed the same or superior efficacy as the fungicide used on conidial germination at all tested concentrations. The MIC50 totally reducing mycelial growth and conidial germination was 6.25 μl/ml. T. peruviana seeds extracts can be exploited in integrated pests management against M. fijiensis.