Basidiospore Germination Research Articles

Sustainable management of blister and grey blight diseases of tea using antagonistic and plant growth promoting microbes in Western Ghats of India

The blister and grey blights are the devastating leaf diseases in tea. The severity of blister and grey blight diseases in tea ranged from 22% to 68% and 11.41%–57.02%, respectively in a survey conducted in Southern India during 2022–2023. The morphological and molecular identifications of pathogens revealed that Exobasidium vexans and Neopestalotiopsis clavispora were responsible for causing blister and grey blights on tea in southern India. The conventional management strategy with foliar spray of synthetic fungicides result in fungicidal residue in tea causes environmental pollution and health hazards to human. To overcome this the current study aims to manage the blister and grey blight diseases of tea through the microbial biocontrol agents. The bacterial biocontrol agents viz., Bacillus subtilis (BBV57), Bacillus amyloliquefaciens (TNAU), Pseudomonas fluorescens (Pf1) and the fungal antagonist, Trichoderma asperellum (TV 1) were evaluated for their efficacy against the both blight diseases. All the bio agents have significantly reduced the basidiospore germination and the mycelial growth of E. vexans and N. clavispora, respectively. Among the bio agents, B. amyloliquefaciens registered 88.36% reduction in the germination of E. vexans basidiospore and 85.56% of mycelial growth reduction of N. clavispora in vitro (P < 0.05). Under field conditions the combined soil and foliar application of bacterial antagonists viz., B. subtilis, B. amyloliquefaciens and P. fluorescens each with 1 ˟ 1011 CFU/ml @ 5% concentration at 7 days interval significantly reduced the blister blight incidence with 53.53% & 51.53% reduction over control and 51.53% & 43.65% of reduction over initial during first and second year, respectively. The combined application also reduced the grey blight incidence with 53.53% & 52.00% reduction over control and 56.81% & 47.65% of reduction over initial during first and second year, respectively. This treatments with bioagents were almost comparable with synthetic treatments, such as copper oxy chloride (0.25%) and hexaconazole (0.25%) with 64.63% and 64.76% reduction of blister and grey blight reduction in first year; 59.74%, 51.11% reduction in second year over control (P < 0.05), respectively. Thus, the application of B. subtilis, B. amyloliquefaciens and P. fluorescens can be recommended for the sustainable management of tea blister and grey blight diseases in tea gardens.

In vitro and in planta investigation of succinate dehydrogenase inhibitor resistance conferred by target-site amino acid substitutions in Puccinia horiana, chrysanthemum white rust.

Resistance to succinate dehydrogenase inhibitor (SDHI) fungicides has been reported in some rust fungi within Pucciniales. However, measuring the resistance factors conferred by a specific substitution at the target site is difficult for most species because of the difficulty in performing in vitro experiments and the complexity of the binuclear state in these obligate parasites. We focused on Puccinia horiana because it easily forms homozygous basidiospores that are sensitive to SDHIs during in vitro germination, whereas the uredospores of other rust fungi are less sensitive. We identified two substitutions, SdhC-I88F and SdhD-C125Y, that drive SDHI resistance in Pu. horiana. Using basidiospore germination inhibition tests, we measured the resistance factors for six SDHI fungicides in Pu. horiana isolates harboring SdhC-I88F substitutions, wherein orthologous substitutions were most frequently observed in SDHI-resistant Pucciniales, such as soybean rust (Phakopsora pachyrhizi). The resistance factors were high for penthiopyrad and benzovindiflupyr (>150), moderate for oxycarboxin and inpyrfluxam (10-30), and low for mepronil and fluxapyroxad (3-10). The most potent SDHI against SdhC-I88F-harboring isolates was inpyrfluxam, with a half-maximal effective concentration (EC50) of 0.0082 mg L-1 owing to its high intrinsic activity. SdhD-C125Y played a minor, but significant role in increasing the resistance factors (one- to tenfold increases), depending on the individual SDHIs. This study is the first to use basidiospore germination inhibitory tests to quantify the resistance factors for SDHI-resistant Pucciniales. Owing to its homozygous binucleate nature and the high availability of basidiospores, Pu. horiana is useful for investigating SDHI resistance in Pucciniales. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of Temperature, Moisture, and Ultraviolet Light on Germination, Infection, and Survival of Gymnosporangium yamadae Teliospores and Basidiospores.

Apple rust caused by Gymnosporangium yamadae is a significant disease in China's main apple production areas. We evaluated the effects of temperature, moisture, and ultraviolet (UV) light on the germination, infection, and survival of teliospore horns and basidiospores under artificially controlled environmental conditions. The temperature required for the germination and infection of teliospores and basidiospores of G. yamadae ranged from 5 to 25°C, with an optimum temperature of approximately 17°C. The teliospore horns germinated after soaking in distilled water for 5 min and required at least 2.3 h of development to produce basidiospores under the most favorable conditions. The basidiospores germinated only in free water and produced germ tubes 0.8 h after being placed in the water. The half-life of the basidiospore was 72.5 h in the dark and only 9.5 h when exposed to intense UV light. The basidiospores inoculated on the host leaves required at least 2.3 h of water exposure to cause rust lesions. A revised Weibull model could describe the relationships between the germination and infection of teliospore horns and basidiospores with temperature and wetness duration. Collectively, these results can serve as a valuable guide for developing a model to predict future apple rust epidemics and establish a method for effective control strategies.

Effect of sodium chloride on basidiospore germination and vegetative mycelial growth of the ectomycorrhizal fungus Rhizopogon roseolus.

Rhizopogon roseolus is a basidiomycetous ectomycorrhizal fungus that inhabits mainly coastal areas. Understanding the response of this fungus to salinity at each stage of its life cycle will lead to elucidation of the strategies for its propagation. This study examined the effect of sodium chloride (NaCl) on basidiospore germination and mycelial growth of both homokaryotic and heterokaryotic strains of R. roseolus, on nutrient agar media with varying concentrations of NaCl (0, 50, 150, and 300 mM). Regardless of the presence of NaCl, R. roseolus basidiospores germinated and the germlings grew, forming compatible fusions. In addition, all multispore strains, including homokaryons and heterokaryons, grew under these NaCl conditions. Most of these strains had an effective concentration inhibiting mycelial growth by 50% value greater than 300 mM of NaCl. These results indicate that R. roseolus can germinate, grow, and mate in the presence of NaCl, allowing it to propagate in saline habitats.

Amino acid substitutions responsible for different QoI and SDHI sensitivity patterns in Puccinia horiana, the causal agent of chrysanthemum white rust

AbstractQuinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) are major groups of agricultural fungicides. However, resistance to some of these fungicides has been reported in a Japanese population of Puccinia horiana, the causal agent of chrysanthemum white rust disease. Because their mechanisms are not well understood, we investigated the existence of mutations in QoI and SDHI target protein‐encoding genes. Eight out of nine isolates from cultivated chrysanthemum carried L275F and L299F amino acid substitutions in cytochrome b, the target protein of QoIs. These isolates showed 23‐ and 17‐fold higher EC50 values for the QoI fungicides azoxystrobin and kresoxim‐methyl, respectively, in basidiospore germination inhibitory tests, while they were hypersensitive to another QoI, famoxadone. All nine isolates were resistant to SDHI oxycarboxin and carried the I88F substitution in SdhC. This substitution was orthologous to the SdhC‐I86F substitution found in some Brazilian isolates of the soybean rust fungus, Phakopsora pachyrhizi, showing reduced sensitivity to some SDHIs. Although the rarity of wild‐type sensitive isolates, the subsequent limited number of comparisons between wild types and mutants, and a difficulty in applying reverse genetic analysis to this obligate parasite, are obstacles in making definitive conclusions, L275F and L299F in cytochrome b and SdhC‐I88F are suspected to be responsible for the different patterns of sensitivity to QoI and for oxycarboxin‐resistance in P. horiana, respectively.

Genome-wide mRNA and miRNA analysis in the early stages of germ tube outgrowth in Coprinopsis cinerea

Coprinopsis cinerea is a model mushroom-forming basidiomycete which produces basidiospores during sexual reproduction. This fungus is widely used to study fruiting body formation and development. Molecular mechanisms controlling its growth from vegetative mycelium to multicellular mature fruiting body have been studied extensively. However, little is known about the underlying biological processes during germ tube outgrowth or the transition from basidiospores to multinucleate hyphae. To better understand sexual spore germination in fungi, here we examined the time-dependent cellular events at resting, germinating and fully germinated basidiospores of C. cinerea by genome-wide transcriptional and post-transcriptional analyses and by carbohydrate composition analysis. Our results revealed a high demand of protein degradation, and biosynthesis of various compounds at the early stage of basidiospore gemination and dynamic changes of carbohydrate metabolism throughout the germination process. Seven microRNA-like RNAs (milRNAs) were identified in the resting basidiospores of C. cinerea, six of which were basidiospore-specific. Glycogen and trehalose were shown to be the carbon sources supporting the initiation of germ tube outgrowth. One basidiospore-specific milRNA, cci-milR-37, was found to be a potential regulator of glycogen metabolic pathways related to vegetative hyphal growth. Our results demonstrated the mRNA and miRNA-mediated regulation on energy production, protein and carbohydrate metabolisms at the early developmental stages of germ tube to form totipotent hyphae. To our knowledge, this is the first study to show the roles of miRNAs in mushroom basidiospore germination and out-growth.

Cultural conditions for basidiospore germination of Lentinus swartzii and Lentinus strigosus and their morphogenesis

Cultural conditions for basidiospore germination of Lentinus swartzii and Lentinus strigosus and their morphogenesis

Development of technique to promote basidiospore germination and single spore isolation of Coprinus comatus

Development of technique to promote basidiospore germination and single spore isolation of <i>Coprinus comatus</i>

Hydrosoluble phylloplane components of Theobroma cacao modulate the metabolism of Moniliophthora perniciosa spores during germination

The surface of plants forms a defense barrier that directly inhibits the first point of contact of microorganisms with the host. To understand this defense mechanism in Moniliophthora perniciosa interaction with Theobroma cacao cv Catongo, the aim of this study was to compare the changes in protein expression in basidiospores of the fungus M. perniciosa in response the leaf water washes (LWW) of two contrasting cocoa varieties for resistance to witches’ broom disease. A total of 8.1 × 108 basidiospores were used for each treatment containing washed leaf material. Germinated basidiospores in the absence of LWW were used as control. The proteomic analysis was performed by the 2D-PAGE technique combined with mass spectrometry (MS). Protein extraction was based on the SDS-dense method followed by sonication for cell disruption and phenol extraction. Sixty-four proteins had accumulation of variation when compared to the control (no LWW). Proteins were identified associated with energy (ATP synthase) and protein (BiP) metabolism, whose accumulation was reduced by basidiospores germinated in leaf wash from Catongo cocoa. The reduction in ATP synthase of the basidiospores germinated the Catongo LWW suggests a shift from aerobic to fermentative metabolism of the fungus in response to components of the LWW. Furthermore, proteins involved in virulence were identified along with fungal resistance to polyketide cyclase, glycoside hydrolase, multidrug transporter protein (SFM) and proteins related to oxidative stress and fermentation, such as catalase A and alcohol dehydrogenase (ADH). The data showed an effect of cocoa phylloplane substances on the germination of fungal basidiospores through differences in protein expression patterns in the presence of LWW of the CCN51 and Catongo genotypes. These results may reveal mechanisms of resistance, host susceptibility and pathogen virulence.

Pear rust: peculiarities of pathogen biology measures for control and profilactic

Pear rust: peculiarities of pathogen biology measures for control and profilactic

Improvement of basidiospore germination and mycelial growth of the ectomycorrhizal mushroom Rhizopogon roseolus in axenic conditions

Improvement of basidiospore germination and mycelial growth of the ectomycorrhizal mushroom Rhizopogon roseolus in axenic conditions

Characteristics, transcriptional patterns and possible physiological significance of glycoside hydrolase family 16 members in Coprinopsis cinerea.

The glycoside hydrolase (GH) 16 family of Coprinopsis cinerea includes 15 members distributed in four subgroups (A1, A2, B and D) by phylogenetic analysis. The expression patterns match well with the requirement of wall-softening in the germination of basidiospores, hyphal growth and branching, primordium formation, stipe elongation, pileus expansion and autolysis. Remarkably, expression levels of different GH16 members varied with different morphogenetic events. Like orthologs of Aspergillus fumigatus GH16 glucanases (ENG2-5), which were expressed in the dormant conidia and conidiogenesis, and essential for segregation of conidia, some members such as ENG in the subgroup A1 in C. cinerea were also predominantly expressed in dormant basidiospores, primordia and maturing pilei during basidiosporogenesis. In contrast, other members in subgroup A2, subgroup B or D were dominantly expressed in the germinating basidiospores, the growing mycelia, and the elongating stipes. We did not find the members of the GH81 or GH55 family in C. cinerea genome, which was different from A. fumigatus. However, C. cinerea contains an extra three subgroups (A2, B and D) compared with A. fumigatus. These extra subgroups of GH16 family members may function as those endo-β-1,3-glucanases belonging to other GH families in the development and growth of C. cinerea.

Establishment of monokaryotic and dikaryotic isolates of Hedgehog mushrooms (Hydnum repandum and related species) from basidiospores

Abstract Hydnum repandum and its relatives are gourmet edible ectomycorrhizal mushrooms. However, no reliable pure cultures have been reported in this genus. Here, we report for the first time the successful isolation of mycelial strains from basidiospores in the genus Hydnum. Basidiospores obtained from basidioma samples were aseptically inoculated onto modified Norkran's C (MNC) medium, MNC containing n-butyric acid (n-MNC), or MNC with gellan gum instead of agar (G-MNC). Although basidiospore germination was observed in most samples, the isolation rate was higher from MNC (91.7%) and G-MNC (93.8%) than from n-MNC (36.4%). Most established isolates were monokaryotic and lacked a clamp connection, but three were dikaryotic and had clamp connections. Established isolates were identified by molecular phylogenetic analysis of the internal transcribed spacer region of the rRNA gene. These results suggest that basidiospores can be used to establish monokaryotic and dikaryotic isolates of Hydnum species.

The effects of environmental and genetic factors on the germination of basidiospores in the Cryptococcus gattii species complex

Natural and artificial hybridization has been frequently reported among divergent lineages within and between the two closely related human pathogenic fungi Cryptococcus gattii species complex and Cryptococcus neoformans species complex. However, the biological effects of such hybridization are not well known. Here we used five strains of the C. neoformans species complex and twelve strains of the C. gattii species complex to investigate the potential effects of selected environmental and genetic factors on the germination of their basidiospores from 29 crosses. We found that the germination rates varied widely among crosses and environmental conditions, ranging from 0% to 98%. Overall, the two examined media showed relatively little difference on spore germination while temperature effects were notable, with the high temperature (37 °C) having an overall deleterious effect on spore germination. Within the C. gattii species complex, one intra-lineage VGIII × VGIII cross had the highest germination rates among all crosses at all six tested environmental conditions. Our analyses indicate significant genetic, environmental, and genotype-environment interaction effects on the germination of basidiospores within the C. gattii species complex.

Study of sodium 3-hydroxycoumarin as inhibitors in vitro, in vivo and in silico of Moniliophthora perniciosa fungus

The inhibition of the fungus Moniliophthora perniciosa, which is the causal agent of witches’ broom disease in Theobroma cacao L., is the main focus of the present study. The use of phytoalexins has provided disease control that is as efficient as control with the use of fungicides, with the advantage of not harming humans and the environment. In this sense, the objective was to evaluate the action of 3-hydroxycoumarin compared with the standard defence activator acibenzolar-S-methyl (Bion® 500 WG) and systemic fungicide tebuconazole (Folicur® 200 CE), and study the possible mechanisms of action. Initially, basidiospore germination inhibition assays (in vitro) were performed using four different concentrations of 3-hydroxycoumarin. The result was 100% inhibition at the concentration of 1000 ppm. Thereafter, this substance was used in four different treatments (in vivo) of cacao seedlings of the SIC-23 genotype, with regard to the order of application. The data analysis showed greater inhibition when 3-hydroxycoumarin was applied after inoculation (TAI test), and suggested a curative effect on Theobroma cacao seedlings. In silico, it was used the enzyme chitin synthase because this action as key enzyme in chitin biosynthetic pathway. The in silico tests show that 3-hydroxycoumarin inhibited the production of chitin synthase by the fungus. Therefore, the present study identifies a potential inhibitor of M. perniciosa and suggests the best application methodology.

The rust fungus Puccinia arechavaletae, a potential biological control agent of balloon vine (Cardiospermum grandiflorum) in South Africa. I: Biology

Studies on the rust fungus Puccinia arechavaletae, were conducted to determine its potential for use as a classical biological control agent against the alien environmental weed Cardiospermum grandiflorum in South Africa. The production of basidiospores is a crucial stage in the life cycle of this microcyclic rust species, and was determined to optimally occur at 20 °C, under high relative humidity (above 90%), in the presence of free water. The first basidiospores were released from mature telia after being incubated for 4 h under optimum conditions, reaching a peak of production after 24 h. Germination of basidiospores optimally occurred at 20 °C, and they were extremely vulnerable to desiccation. Germinating basidiospores directly penetrated epidermal cells of South African C. grandiflorum within which a vesicle formed, followed by rapid radial colonization of the leaf tissue by fungal mycelium. The first symptoms observed were small chlorotic spots on the leaves from 14 days onwards, with telial pustules appearing between 14 and 21 days post inoculation. The rust P. arechavaletae was shown to be highly pathogenic towards South African C. grandiflorum, causing severe disease symptoms with a detrimental effect on plant health and vigor.

Ganoderma basidiospore germination responses as affected by spore density, temperature and nutrient media

The present study investigated the axenic basidiospore germination of Ganoderma australe, G. mastoporum and G. philippii at three spore densities in five nutrient media encompassing a range of carbohydrate complexity, in combination with sawdust and/or ethanol as medium additives. Five incubation temperatures ranging from 10 to 35 °C were used, totalling 300 treatment combinations. AWeibull model, with the asymptote occurring at 50 to 75 h, provided good fit to percentage spore germination data at each treatment combination for all three Ganoderma species. Ganoderma australe and G. mastoporum basidiospores germinated on all media, whereas G. philippii basidiospores required media that contained 2% ethanol. The best medium for G. australe and G. mastoporum basidiospore germination was rice dextrose agar with amixture of Eucalyptus and Acacia sawdust, whereas for G. philippii it was 1% malt extract agar plus ethanol, with or without sawdust. Spore density was also critical to achieving the best germination rate, with ~400 spores/cm2 optimal for all three species. As Ganoderma root rot disease affects commercial Acacia mangium and Eucalyptus pellita plantations, the greater understanding of basidiospore germination gained from the current study should assist in developing strategies to contain the dispersal and spread of root rot in Indonesia and other southeast Asian countries where these species have been planted.

Effect of temperature, leaf wetness and the developmental stage of host tissue on infection of Acacia mearnsii by Uromycladium acaciae (Pucciniales)

Uromycladium acaciae causes a serious rust disease in plantations of non-native Acacia mearnsii (black wattle) in South Africa. Little is known about the biology of U. acaciae, making disease control difficult. Germination studies and artificial inoculations were conducted to identify the optimal environmental conditions for infection by U. acaciae. Germination of teliospores, basidiospores, and urediniospores was assessed at seven temperatures, with or without light. The effect of temperature on infection was also assessed. As was the effect of dew period length on germination of teliospores, production of basidiospores and infection. Teliospores and urediniospores germinated between 5 and 30 °C, with an optimum at 15–25 °C. Basidiospores were produced and germinated at temperatures between 5 and 25 °C, with an optimum at 15–20 °C. The optimum temperature for infection by basidiospores was 15–20 °C. All spore types germinated in 6–24 h under optimal conditions. However, production of basidiospores was severely reduced if teliospore germination was interrupted by dry periods, even if teliospores were re-wetted. Symptoms and telia developed on only one plant exposed to a dew period of less than 12 h, with a dew period of 48 h found to be optimal. Artificial inoculation experiments showed that U. acaciae was only able to infect young, growing tissues. Results of this research can be used to develop an artificial inoculation protocol for resistance screening and in disease risk modelling and forecasting.

Genetic and environmental influences on the germination of basidiospores in the Cryptococcus neoformans species complex

In basidiomycetous fungi, the viability of basidiospores is an important component of sexual fitness. However, relatively little is known about the genetic and environmental factors influencing basidiospore germination. In this study, we used human opportunistic yeast pathogens, Cryptococcus neoformans and Cryptococcus deneoformans, as models to investigate the potential effects of selected genetic and environmental factors on basidiospore germination. A total of five strains with known genome structure were used to construct six crosses, three of which were between strains within the same species, while the remaining three were hybrid crosses between C. neoformans and C. deneoformans. Offspring from these crosses were incubated on two media (a nutrient-limiting and a nutrient-rich) and three temperatures (23 °C, 30 °C, and 37 °C). In general, spores from intra-specific crosses had greater germination rates than those from inter-specific crosses. Of the two environmental factors, temperature showed a greater influence than nutrient medium, with the 37 °C environment yielding lower germination rates than at 23 °C and 30 °C environments in most crosses. Furthermore, there were notable interaction effects between environmental factors and parental strains or strain pairs on basidiospore germination. We discuss the implications of these results on pathogenesis and speciation in this human fungal pathogen.

Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin.

Zeaxanthin is an essential nutrient for prevention of macular degeneration. However, it is limited in our diet. For the production of zeaxanthin, we have engineered zeaxanthin synthesis into a carotenoid mutant of Xanthophyllomyces dendrorhous which is blocked in astaxanthin synthesis and accumulates β-carotene instead. Two strategies were followed to reach high-yield zeaxanthin synthesis. Total carotenoid synthesis was increased by over-expression of genes HMGR, crtE, and crtYB encoding for limiting enzymes in the pathway leading to and into carotenoid biosynthesis. Then bacterial genes crtZ were used to extend the pathway from β-carotene to zeaxanthin in this mutant. The increase of total carotenoids and the formation of zeaxanthin is dependent on the number of gene copies of crtYB and crtZ integrated into the X. dendrorhous upon transformation. The highest zeaxanthin content around 500μg/g dw was reached by shaking flask cultures after codon optimization of crtZ for Xanthophyllomyces. Stabilization of carotenoid and zeaxanthin formation in the final transformant in the absence of selection agents was achieved after passing through a sexual cycle and germination of basidiospores. The values for the transformant before and after stabilization were very similar resembling about 70% of total carotenoids and corresponding to a conversion rate of 80% for hydroxylation of β-carotene to zeaxanthin. The stabilized transformant allowed experimental small-scale fermentation yielding X. dendrorhous cells with a zeaxanthin content similar to the shaking flask cultures. Our result demonstrates the potential of X. dendrorhous for its development as a zeaxanthin producer and its suitability for large-scale fermentation.