Sporulation Capacity Research Articles

Genomic epidemiology of Clostridioides difficile Sequence Type 35 reveals intraspecies and interspecies clonal transmission

ABSTRACT Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.

Genetic structure and pyrimethanil resistance of Botrytis spp. causing gray mold on strawberry from greenhouses in Zhejiang, China

Gray mold caused by Botrytis spp. is a common disease on various hosts, including strawberries. In this study, we obtained 59 Botrytis isolates from strawberries from greenhouses in Zhejiang Province, China. Identification of the sampled isolates at species level was performed by a duplex PCR assay method, the result showed that, in Zhejiang, gray mold on strawberry fruits is caused by a complex of Botrytis groups including B. cinerea group N (47.5 %) and B. cinerea group S (52.5 %). The sensitivities of all Botrytis isolates to pyrimethanil were determined based on discriminatory dose method on L-asparagine-based agar medium plate. Our results showed that the isolates obtained from the greenhouses with continuous use of pyrimethanil developed severe resistance to pyrimethanil, and the resistance frequencies of B. cinerea group N and group S isolates were 89.3 % and 41.9 %, respectively. By sequencing, four different resistance-related point mutations were identified in 38 Botrytis isolates: Bcpos5L412F (16 isolates, 42.1 %), Bcpos5L412V (14 isolates, 36.8 %), Bcmdl1E407K (2 isolates, 5.3 %), and Bcpos5L412S⸱Bcmdl1E407K (1 isolate, 2.6 %). The exogenous addition of methionine could not completely alter the resistance of Botrytis isolates to pyrimethanil. In this study, the pyrimethanil resistance in Botrytis isolates was steadily inherited, and compared to the pyrimethanil-sensitive isolates, the resistant mutants exhibited good fitness in sporulation capacity, spore germination rate, and virulence on strawberries. In conclusion, our results provided a description of the genetic structure of Botrytis groups complex on strawberry fruits and reminded growers to focus on the stable pyrimethanil resistance in Botrytis isolates, caused by point mutations in BcPos5 and BcMdl1.

A Biological Comparison of Three Colletotrichum Species Associated with Alfalfa Anthracnose in Northern China.

Anthracnose caused by various species of Colletotrichum is one of the most prevalent diseases in alfalfa worldwide that not only reduces forage yields but also severely compromises forage quality. A comprehensive survey was conducted in 2020 in the main production regions of northern China. The survey results showed that alfalfa anthracnose is prevalent in northern China, with the disease incidence ranging from 9% to 45% and the disease index from 5 to 17 (maximum possible score: 100). In total, 24 isolates were collected and identified as three Colletotrichum species (C. trifolii, C. truncatum and C. americae-borealis) based on morphological characteristics and phylogenetic analysis (combined sequences ITS, HIS3, ACT and GAPDH). The three species displayed remarkable environmental adaptability, exhibiting a capacity for growth, sporulation and conidial germination in temperatures ranging from 4 to 35 °C and in different nutrient conditions. Pathogenicity assays showed that C. trifolii was more virulent than the other two species, although the growth vigor (in terms of colony diameter, sporulation and conidial germination) of C. truncatum was the greatest.

Resistance of Alicyclobacillus acidoterrestris spores to atmospheric cold plasma: Insights from sporulation temperature and mechanism analysis

Environmental conditions during sporulation, such as temperature, pH value, and mineral content, can significantly influence the formation production and resistance of spores. In this study, we investigated the sporulation capacity and resistance of Alicyclobacillus acidoterrestris (AAT) spores obtained at different temperatures (25 °C, 35 °C, 45 °C) to Dielectric Barrier Discharge Atmospheric cold plasma (DBD-ACP, 20 and 30 kV for 2–8 min). The results indicated that the efficiency of sporulation was highest at the optimum temperature (45 °C), deviation from optimal temperature would have a negative effect on sporulation. Otherwise, sporicidal efficiency of DBD-ACP increased with longer treatment time and lower sporulation temperatures, suggesting that AAT spores exhibit weaker resistance to DBD-ACP at lower sporulation temperature. Results from the release of DPA, and electron microscopic observations confirmed that DBD-ACP caused disruption of the cortex and membrane structure of AAT spores, leading to the release of intracellular contents and subsequent spore's death. Further analysis through proteomics revealed intriguing insights into the differences between spores formed at 25 °C and those at 45 °C. Specifically, spores formed at 25 °C exhibited lower expression levels of proteins related to sporulation, intracellular energy metabolism, membrane transport, and significant down-regulation of proteins involved in peptidoglycan and spore coat formation compared to those at 45 °C. These alterations likely contribute to the development of a thinner and looser spore structure, rendering it less effective in defending, i.e. less resistant to DBD-ACP treatment. Industrial relevanceAAT spores' resistance to pasteurization and persistence in NFC juices impact juice quality and safety. This study investigates the resistance of AAT spore to DBD-ACP treatment under different sporulation temperatures. Insights into sporicidal behavior of DBD-ACP and the effect of sporulation temperature on resistance provide essential knowledge for optimizing juice processing and enhancing microbial safety. The findings offer practical guidance for the juice industry to develop improved preservation strategies, ensuring longer shelf life and consumer satisfaction.

Screening, characterization and optimization for synergistic interaction of potential dichlorodiphenyltrichloroethane degrading fungi isolated from agro-industrial effluent and farm soil

Dichlorodiphenyltrichloroethane (DDT) is a recalcitrant synthetic chemical that threatens the environment. Despite being banned in most industrialized nations, DDT is still used as a pesticide to fight malaria and agricultural pests. The main objective of this study is to screen, characterize, and evaluate potential DDT-degrading fungi and their synergetic interaction effects for mycoremediation purpose. The soil and effluent samples were collected from Ziway, Koka, and Addis Ababa. Fungal isolation and screening were performed using a serial dilution on potato dextrose agar growth media. Matrix-Assisted Laser Desorption, Ionization, and Time of Flight Mass Spectrometry technology was used for fungal identification and the selected isolates AS1 and T1 were identified as Aspergillus niger and Trichoderma koningii. Fungal biomass production and sporulation capacity were examined and optimized using a Box-Behnken experimental design. The potential DDT-tolerant fungi were studied based on growth factor optimization. The optimization results revealed that the co-inoculated isolates AS1T1 had a maximum biomass (1.01 ± 0.16g) and spore count (5.74 ± 0.37 log spore/mL) and were selected as possible DDT-degrading fungi. Gas Chromatograph-Electron Capture Detector technology was used for the DDT degradation study. Its analysis confirmed that fungal-co-cultured Aspergillus niger and Trichoderma koningii in DDT-amended liquid medium were able to degrade DDT into its metabolites (DDE and DDD). The results also revealed that 99.5–99.99% of DDT and its metabolites degraded from initial concentrations of 1750, 3500, 5250, and 7000 ppm. The co-inoculated fungi Aspergillus niger and Trichoderma koningii are promising candidates for the removal of DDT and its metabolites from polluted environments.

Blackleg: A Review of the Agent and Management of the Disease in Brazil.

The genus Clostridium is an important group of pathogenic and nonpathogenic Gram-positive anaerobic bacteria with a sporulation capacity and wide distribution in different environments, including the gastrointestinal tracts of healthy and diseased animals and humans. Among the pathogenic species of the genus, Clostridium chauvoei stands out as a histotoxic agent. It causes significant myonecrosis such as blackleg, a disease with high lethality, especially in young cattle, and is responsible for significant livestock losses worldwide. The pathogenicity of the disease is complex and has not yet been fully elucidated. Current hypotheses cover processes from the initial absorption to the transport and deposition of the agent in the affected tissues. The virulence factors of C. chauvoei have been divided into somatic and flagellar antigens and soluble antigens/toxins, which are the main antigens used in vaccines against blackleg in Brazil and worldwide. This review provides important information on the first and current approaches to the agent C. chauvoei and its virulence factors as well as a compilation of data on Brazilian studies related to blackleg.

Risk Factors and Environmental Preventive Actions for Aspergillosis in Patients with Hematological Malignancies.

(1) Background: Aspergillus spp. is a widely distributed filamentous fungus in the environment due to its high sporulation capacity. Currently, invasive aspergillosis (IA) is the most common invasive fungal infection in patients with hematologic malignancies, with high rates of mortality and morbidity. The multifactorial nature of the disease requires appropriate risk stratification to enable the most appropriate preventive measures to be adapted and implemented according to the characteristics of the patient. In this sense, the present research aims to identify recent risk factors and environmental control measures against invasive aspergillosis to establish preventive actions to reduce the incidence of invasive aspergillosis in hospitals. (2) Methods: We conducted a qualitative systematic review of the scientific literature on environmental risk factors and preventive measures for invasive aspergillosis in patients with hematologic malignancies. The Medline, Cochrane, and Scopus databases were consulted, following the PRISMA and STROBE guidelines. (3) Results: Adequate implementation of environmental control measures is presented as the most efficient intervention in terms of prevention to decrease the incidence of invasive aspergillosis in hospitals. Neutropenia, fungal contamination, insufficient environmental control measures in hospital and home settings, length of hospital stay, and anemia, are identified as independent risk factors. We show that HEPA, LAF, and Plasmair® systems are suitable methods to reduce the concentration of airborne fungal spores. Antifungal prophylaxis did not significantly influence IA reduction in our study. (4) Conclusions: Proper professional training and environmental control measures in hospitals are essential for the prevention of invasive aspergillosis. We should optimize risk stratification for patients with hematologic malignancies. Antifungal prophylaxis should be complementary to environmental control measures and should never be substituted for the latter. Studies should also be undertaken to evaluate the efficiency of environmental control measures against IA at patients' homes.

The sprT Gene of Bacillus velezensis FZB42 Is Involved in Biofilm Formation and Bacilysin Production

Bacillus velezensis FZB42, a representative strain of plant-growth-promoting rhizobacteria (PGPR), can form robust biofilm and produce multiple antibiotics against a wild range of phytopathogens. In this study, we observed different biofilm morphology of the mutant Y4, derived from a TnYLB-1 transposon insertion library of B. velezensis FZB42. We identified that the transposon was inserted into the sprT gene in Y4. Our bioinformatics analysis revealed that the SprT protein is an unstable hydrophilic protein located in the cytoplasm. It is highly conserved in Bacillus species and predicted to function as a metalloprotease by binding zinc ions. We also demonstrated that ΔsprT significantly reduced the swarming ability of FZB42 by ~5-fold and sporulation capacity by ~25-fold. In addition, the antagonistic experiments showed that, compared to the wild type, the ΔsprT strain exhibited significantly reduced inhibition against Staphylococcus aureus ATCC-9144 and Phytophthora sojae, indicating that the inactivation of sprT led to decreased production of the antibiotic bacilysin. The HPLC-MS analysis confirmed that bacilysin was indeed decreased in the ΔsprT strain, and qPCR analysis revealed that ΔsprT down-regulated the expression of the genes for bacilysin biosynthesis. Our results suggest that the sprT gene plays a regulatory role in multiple characteristics of B. velezensis FZB42, including biofilm formation, swarming, sporulation, and antibiotic production.

Bacillus megaterium RTS1 enhances resistance of Lycopersicon esculentum to salinity stress through the improvement of antioxidant defenses

Background and Objectives: Plant growth-promoting bacteria (PGPB) may reduce the negative effects of salinity stress. The aim of this study was to optimize Bacillus megaterium RTS1 and characterize the effect of the PGPB on the physiolog- ical characteristics of tomato (Lycopersicon esculentum).
 Materials and Methods: The Central composite design (CCD) of response surface methodology (RSM) was used to opti- mize Bacillus megaterium RTS1 to produce maximum cell biomass and spores. Then the effect of the PGPB on the physi- ological characteristics of tomato (Lycopersicon esculentum), including membrane stability, leaf relative water content per- centage, anthocyanin and carotenoids content, chlorophyll photosynthetic parameters, sugar and starch level, superoxide anion and antioxidant activity under salt stress conditions. The NFB medium was inoculated with 5% bacterial culture and the fermentation was carried out in a 10-lit fermenter.
 Results: After optimization, the amount of cell biomass by the model was 9.45 log10 CFUs/mL, which showed a 1.2-fold increase compared to the non-optimized medium. Usage of bacteria under the optimal conditions of the culture medium may increase the stability of the membrane and improve the relative water content. Bacteria were able to prevent the excessive increase of anthocyanins. Oxidative stress led to an increase in the content of chlorophyll a, while causing the degradation of chlorophyll b. Bacterial inoculation led to an increase in the level of sugar and starch compared to the control. PGPB showed an increasing effect on the amount of superoxide anion production and caused a significant increase in the antioxidant activity under salinity stress conditions.
 Conclusion: The PGPB can be a promising way to boost physiological characteristics of tomato plant under salinity stress. Also, sporulation capacity of Bacillus megaterium with high bacterial cell density in fermenter produce a sustainable product for tomato plants.

Cellular communication and fusion regulate cell fusion, trap morphogenesis, conidiation, and secondary metabolism in Arthrobotrys oligospora

Signal-mediated cell fusion is vital for colony development in filamentous fungi. Arthrobotrys oligospora is a representative nematode-trapping (NT) fungus that produces adhesive networks (traps) to capture nematodes. Here, we characterized Aoadv-1, Aoso, Aoham-6, and Aoham-5 of A. oligospora, homologs of proteins involved in cellular communication and fusion in the model fungus Neurospora crassa. The deletion of four genes resulted in the complete loss of cell fusion, and traps produced by mutants did not close to form mycelial rings but were still capable of capturing nematodes. The absence of these genes inhibits aerial mycelial extension, slows colony growth, and increases mycelial branching. In addition, the mutants showed reduced sporulation capacity and tolerance to oxidative stress, increased sensitivity to SDS, and disturbed lipid droplet accumulation and autophagy. In addition, transcriptome and metabolomic analyses suggested that Aoadv-1 and Aoso are involved in multiple cellular processes and secondary metabolism. Our results revealed that Aoadv-1, Aoso, Aoham-6, and Aoham-5 regulate mycelial growth and trap morphogenesis through cell fusion, which contributed to elucidating the molecular mechanisms of cellular communication regulating mycelial development and trap morphogenesis in NT fungi.

Molecular Identification, Effector genes and Virulence of Isolates of <i>Parastagonospora nodorum</i> from Altai Krai (Russia)

Septoria blotches represent some of the most harmful wheat diseases that encompass all grain-producing countries worldwide. They are caused mainly by fungi of the genera Parastagonospora and Zymoseptoria. The aims of the research were to analyze the nucleotide sequences of two phylogenetically informative DNA loci, ITS and tub2, of Parastagonospora nodorum isolates from the Altai Krai, to detect the presence of Tox1, Tox3, ToxA effector genes and to study the virulence of the isolates. Microscopic analysis of fragments of affected plant tissue was used for primary identification of the Septoria blotch. The causative agent from the studied plant samples was identified as P. nodorum. Fungal colonies cultivated on potato-glucose agar displayed a well-developed light brown velvety mycelium with dark periphery (mixed type) and numerous pycnidia exhibiting high sporulation capacity. Microbiological diagnostics were complemented by molecular genetic studies. Sequencing of ITS and tub2 loci isolates revealed complete genetic identity of all eight studied monoconidial isolates obtained from different samples. PCR-based detection of tree Tox-genes demonstrated that the only Tox3 was present in the geno-types of 80 tested P. nodorum monoconidial isolates. The ToxA and Tox1 genes were not found in the studied isolates. The virulence of P. nodorum isolates was evaluated in laboratory conditions using an isolated wheat leaves assay. Mixture of four isolates from spring wheat and one isolate from winter wheat, were characterized as virulent. When spring and winter wheat cultivars were infected with two isolates obtained from oats, pathogenic but avirulent properties were detected. The isolate from triticale was avirulent to winter wheat cultivars and virulent to spring wheat cultivars.

Evolution within a given virulence phenotype (pathotype) is driven by changes in aggressiveness: a case study of French wheat leaf rust populations

Plant pathogens are constantly evolving and adapting to their environment, including their host. Virulence alleles emerge, and then increase, and sometimes decrease in frequency within pathogen populations in response to the fluctuating selection pressures imposed by the deployment of resistance genes. In some cases, these strong selection pressures cannot fully explain the evolution observed in pathogen populations. A previous study on the French population of Puccinia triticina, the causal agent of wheat leaf rust, showed that two major pathotypes — groups of isolates with a particular combination of virulences — predominated but then declined over the 2005-2016 period. The relative dynamics and the domination of these two pathotypes — 166 317 0 and 106 314 0 —, relative to the other pathotypes present in the population at a low frequency although compatible, i.e. virulent on several varieties deployed, could not be explained solely by the frequency of Lr genes in the landscape. Within these two pathotypes, we identified two main genotypes that emerged in succession. We assessed three components of aggressiveness — infection efficiency, latency period and sporulation capacity — for 44 isolates representative of the four P. triticina pathotype-genotype combinations. We showed, for both pathotypes, that the more recent genotypes were more aggressive than the older ones. Our findings were highly consistent for the various components of aggressiveness for pathotype 166 317 0 grown on Michigan Amber — a ‘naive’ cultivar never grown in the landscape — or on Apache — a ‘neutral’ cultivar, which does not affect the pathotype frequency in the landscape and therefore was postulated to have no or minor selection effect on the population composition. For pathotype 106 314 0, the most recent genotype had a shorter latency period on several of the cultivars most frequently grown in the landscape, but not on ‘neutral’ and ‘naive’ cultivars. We conclude that the quantitative components of aggressiveness can be significant drivers of evolution in pathogen populations. A gain in aggressiveness stopped the decline in frequency of a pathotype, and subsequently allowed an increase in frequency of this pathotype in the pathogen population, providing evidence that adaptation to a changing varietal landscape not only affects virulence but can also lead to changes in aggressiveness.

Population Dynamics of Phytophthora infestans in Egypt Reveals Clonal Dominance of 23_A1 and Displacement of 13_A2 Clonal Lineage.

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are the most economically important vegetable crops in Egypt and worldwide. The winter crop in Egypt is particularly prone to late blight caused by Phytophthora infestans. A total of 152 P. infestans isolates were isolated from the 2013, 2014, 2016 and 2018 winter crops with 82 isolates from potato, 69 from tomato and one isolate from eggplant (S. melongena L.). All isolates belonged to the A1 mating type with no evidence of A2 or self-fertile strains. The majority of isolates (53%) were sensitive to metalaxyl, 32% were intermediate and 15% were resistant. Variation in aggressiveness between three P. infestans isolates EG-005 (13_A2) and EG-276 (23_A1) from potato, and EG-237 (23_A1) from eggplant was determined on tuber slices and leaflets of 10 potato cultivars. The eggplant isolate EG-237 showed higher sporulation capacity compared with the other tested isolates and was able to infect potato (Lady Rosetta cv) and tomato (Super Strain B cv). The simple sequence repeat (SSR) genotyping data showed that in contrast to our previous work (3-year period 2010-12) in which the proportion of 13_A2 lineage was 35%, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. The data have been submitted into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. The AVR2 and AVR2-like RXLR effector genes were amplified and sequenced. In the avirulent AVR2 gene, only one heterozygous SNP was detected at position 31 in the N terminus in six isolates out of eleven, whereas two heterozygous SNPs were detected at position 29 in the N-terminus and ninety-two in the C- terminus of the AVR2-like gene. This suggests that changes in the previously reported virulence profile of 23_A1 are not related to commercial cultivars carrying the R2 gene. In addition, this is the first report of P. infestans on eggplant in Egypt.

SNARE Protein AoSec22 Orchestrates Mycelial Growth, Vacuole Assembly, Trap Formation, Stress Response, and Secondary Metabolism in Arthrobotrys oligospora.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle trafficking and membrane fusion in eukaryotes and play a vital role in fungal growth, development, and pathogenicity. However, the functions of SNAREs are still largely unknown in nematode-trapping fungi. Arthrobotrys oligospora is a representative species of nematode-trapping fungi that can produce adhesive networks (traps) for nematode predation. In this study, we characterized AoSec22 in A. oligospora, a homolog of the yeast SNARE protein Sec22. Deletion of Aosec22 resulted in remarkable reductions in mycelial growth, the number of nuclei, conidia yield, and trap formation, especially for traps that failed to develop mature three-dimensional networks. Further, absence of Aosec22 impaired fatty acid utilization, autophagy, and stress tolerance; in addition, the vacuoles became small and fragmented in the hyphal cells of the ∆Aosec22 mutant, and large vacuoles failed to form. The reduced sporulation capacity correlated with the transcriptional repression of several sporulation-related genes, and the impaired accumulation of lipid droplets is in line with the transcriptional repression of several genes involved in fatty acid oxidation. Moreover, absence of Aosec22 remarkably impaired secondary metabolism, resulting in 4717 and 1230 compounds upregulated and downregulated in the ∆Aosec22 mutant, respectively. Collectively, our data highlighted that the SNARE protein AoSec22 plays a pleiotropic role in mycelial growth and development, vacuole assembly, lipid metabolism, stress response, and secondary metabolism; in particular, it is required for the proper development of traps in A. oligospora.

Noble Indices for Degree of Host Plant Resistance against Alternaria Blight in Rapeseed-mustard Genotypes

Experiments were conducted at Genetics and Plant Breeding Research Farm of N. D. University of Agriculture and Technology, Kumarganj, Faizabad (U.P.) to evaluate 15 promising genotypes of rapeseed-mustard against Alternaria blight caused by Alternaria brassicae (Berk) Sacc. and A. brassicicola (Schw) Wiltshire. It is the most destructive and major disease problem under eastern Uttar Pradesh in Indian condition causing both the quantitative and qualitative losses. The evaluation criteria for host resistance against the disease considered were the number and size of spot, number of conidia per spot on both the vegetative (leaf) and reproductive (pod) parts were regularly recorded at periodical intervals starting from the disease appearance till the physiological maturity of each genotypes. The indices which were considered for in built resistance were delayed appearance of visible symptoms, minimum number of spot and their size, number of conidia per spot as well as the minimum leaf defoliation. The disease appeared first in genotype T-9 (44 DAS) which was delayed in GSL-5 (53 DAS). The number of spot/10cm2, size, sporulation capacity in terms of conidia per spot varied invariably among the genotypes and significantly in some cases. The appearance of disease symptoms in the form of blighted spots varied invariably (44 to 53 DAS) in different genotypes. The delayed appearance of disease symptoms (53 and 52 DAS), lower number of spot / 10cm2 on leaf (3.07 and 3.65), pods (3.74 and 4.14), smallest spot size on leaf (3.54 and 3.05 cm), pods (1.22 and 1.27 cm2), number of conidia per spot on leaf (1230 and 1630), pods (130 and 150) were visualised in genotype GSL-5 and Pusa Aditya,respectively indicating a certain pattern and may be considered as attributes of disease resistance. Further, this fact was strengthened with minimum leaf defoliation (32.03 and 35.55%), PDI on leaf (12.35 and 14.16), pods (7.73 and 7.93) and ultimately the AUDPC calculated on leaf (486.70 and 551.90) and pods (282.85 and 291.05). Accordingly both the genotypes were grouped in moderately resistant (MR) category. The correlation coefficient (r) among parameters attributing to resistance behaviour worked out proved helpful in drawing the conclusion. Because very limited source of durable resistance are available, it may be suggested to exploit the two genotypes i.e.GSL-5 and Pusa Aditya for breeding programme against Alternaria blight.

English

Coffee berry disease (CBD) caused by Colletotrichum kahawae, is the main disease that affects arabica coffee production in Africa. Isolates of C. kahawae were collected from different altitude in the West Region of Cameroon and characterised for cultural and morphological variations. The results showed a significant variation among C. kahawae isolates related to their morpho-cultural features with respect to altitude. All isolates had cottony mycelium appearance. Four types of colony colours were identified: Grey, whitish, greyish and pinkish. Mycelia growth rate of isolates varied significantly from 4.32 to 7.11 mm/day. Dominant shape of conidia was cylindrical acute and round ends of CBDBHa1, CBDBHa2 and CBDBHa3 isolates from the high altitude, followed by cylindrical round at both ends of isolates CBDBMa1 and CBDBMa2 from medium altitude. Fusiform and reniform conidia shapes were observed with isolates CBDNBa1 and CBDNBa2 from low altitude. Conidia size and sporulation capacity of C. kahawae isolates increased with altitude. Conidia size ranged from 3.82 to 13.46 µm in length and from 1.13 to 1.69 µm in width; while sporulation capacity varied from 169.06 ×104 to 438.36 ×104 conidia/ml. This study highlights some variations in cultural and morphological characteristics of C. kahawae with respect to altitude. Key words: Altitude, coffee berry disease, Colletotrichum kahawae, morpho-cultural characteristics, Coffea arabica.

Sexually-Driven Combinatorial Diversity in Native Saccharomyces Wine Yeasts

Natural diversity represents an inexhaustible source of yeasts for the diversification of wines and the improvement of their properties. In this study, we analysed the genetic diversity of autochthonous Saccharomyces cerevisiae wine yeasts in the Aljarafe of Seville, one of the warmest winemaking regions of Spain. Through multiplex-PCR analysis of five microsatellite markers and RT-PCR determination of the killer genotype, we found 94 different patterns among 150 S. cerevisiae yeast strains isolated from spontaneous fermentation of grape must, thereby representing a highly diverse population. Remarkably, 92% of the isolated strains exhibited high sporulation capacity. Tetrad analysis of sporulating strains rendered a microsatellite marker’s combinatory that mimics patterns observed in the native population, suggesting that the high polymorphism of microsatellite markers found in these wild yeasts might result from sexual reproduction in their natural environment. The identification of unconventional M2/L-A-lus totivirus combinations conferring the killer phenotype also supports this suggestion. One idea behind this study is to determine to what extent the vineyards microbiota in areas with warm climates can provide useful natural yeasts to adapt fermentation processes to the needs imposed by global warming. Analysis of traits of oenological interest in regions potentially affected by global climate changes, such as growth tolerance to ethanol and to sugar stress in the analysed strains, indicated that this broad combinatorial diversity of natural S. cerevisiae yeasts provides a wide range of autochthonous strains with desirable profiles for quality winemaking in warm regions. This combinatorial diversity renders strains with diverse oenological performing abilities. Upon microvinification assays and organoleptic attests, a S. cerevisiae strain with interesting oenological properties has been identified. This result can be considered a successful outcome in industry–academia collaboration.

The Arf-GAP Proteins AoGcs1 and AoGts1 Regulate Mycelial Development, Endocytosis, and Pathogenicity in Arthrobotrys oligospora.

Small GTPases from the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development, endocytosis, and virulence in fungi. Here, we identified two orthologous Arf-GAP proteins, AoGcs1 and AoGts1, in a typical nematode-trapping fungus Arthrobotrys oligospora. The transcription of Aogcs1 and Aogts1 was highly expressed in the sporulation stage. The deletion of Aogcs1 and Aogts1 caused defects in DNA damage, endocytosis, scavenging of reactive oxygen species, lipid droplet storage, mitochondrial activity, autophagy, serine protease activity, and the response to endoplasmic reticulum stress. The combined effects resulted in slow growth, decreased sporulation capacity, increased susceptibility to chemical stressors and heat shock, and decreased pathogenicity of the mutants compared with the wild-type (WT) strain. Although deletion of Aogcs1 and Aogts1 produced similar phenotfypic traits, their roles varied in conidiation and proteolytic activity. The ΔAogts1 mutant showed a remarkable reduction in conidial yield compared with the WT strain but not in proteolytic activity; in contrast, the ΔAogcs1 mutant showed an increase in proteolytic activity but not in sporulation. In addition, the growth of ΔAogcs1 and ΔAogts1 mutants was promoted by rapamycin, and the ΔAogts1 mutant was sensitive to H-89. Collectively, the ΔAogts1 mutant showed a more remarkable difference compared with the WT strain than the ΔAogcs1 mutant. Our study further illustrates the importance of Arf-GAPs in the growth, development, and pathogenicity of nematode-trapping fungi.

Risk of Epidemic Development in Nurseries from Soil Inoculum ofPhytophthora ramorum.

Soilborne inoculum arising from buried, infested leaf debris may contribute to the persistence of Phytophthora ramorum at recurrently positive nurseries. To initiate new epidemics, inoculum must not only survive but also produce sporangia during times conducive to infection at the soil surface. To assess this risk, we performed two year-long experiments in a soil plot at the National Ornamentals Research Site at Dominican University of California. Inoculated rhododendron leaf disks were buried at a depth of 5 or 15 cm in the early summer of 2014 or 2015. Inoculum was baited at the soil surface with noninfested leaf disks (2014 only) and then retrieved to assess pathogen viability and sporulation capacity every 5 weeks. Two 14-week-long trials were conducted in 2016. We were able to consistently culture P. ramorum over all time periods. Soil incubation rapidly reduced the capacity of inoculum to sporulate, especially at 5 cm; however, sporulation capacity increased with the onset of seasonally cooler temperatures. P. ramorum was baited most frequently between November and January, especially from inoculum buried at 5 cm 1 day before the baiting period; in January we also baited P. ramorum from inoculum buried at 15 cm the previous June. We validate prior observations that P. ramorum poses a greater risk after exposure to cooler temperatures and provide evidence that infested leaf debris plays a role in the perpetuation of P. ramorum in nurseries. This work provides novel insights into the survival and epidemic behavior of P. ramorum in nursery soils.

Pseudocercospora griseola, the causal agent of common bean angular leaf spot: Strain characterization and sensitivity to fungicides

AbstractAngular leaf spot (ALS), an important disease of common bean (Phaseolus vulgaris), is caused by the fungus Pseudocercospora griseola. This pathogen has a wide genetic variability and, therefore, poses a challenge to integrated disease management. The use of resistant cultivars is difficult; hence, the application of fungicides has been a common practice in common bean cultivation. P. griseola strains were morphophysiologically characterized and their sensitivity to common fungicides used to control ALS was studied. The strains were evaluated for sporulation capacity and a representative sample of 34 strains was bioassayed to determine their sensitivity to commercial concentrations of five fungicides, namely pyraclostrobin, mancozeb, pyraclostrobin + metconazole, chlorothalonil and tebuconazole. Another sample of 29 strains was studied for conidial germination and dimensions. Sporulation capacity ranged from 0.88 to 27.67 × 104 conidia/ml and germination percentage ranged from 39% to 72%. The large differences among strains suggest a wide genetic variability among the strains. A wide variability in aggressiveness of P. griseola was observed, which has consequences for breeding programmes aimed at resistance. The behaviour of pathogen strains differed in every fungicide evaluated, even in a population that has not been under selection pressure in the field. These results confirm the need for further studies and may guide future research with this pathogen.