Whole genome analyses based on single, field collected spores of the arbuscular mycorrhizal fungus Funneliformis geosporum.

Arbuscular mycorrhizal (AM) fungi are ubiquitous mutualistic symbionts of most terrestrial plants and many complete their lifecycles underground. Whole genome analysis of AM fungi has long been restricted to species and strains that can be maintained under controlled conditions that facilitate collection of biological samples. There is some evidence suggesting that AM fungi can adapt to culture resulting in phenotypic and possibly also genotypic changes in the fungi. In this study, we used field isolated spores of AM fungi and identified them as Funneliformis geosporum based on morphology and phylogenetic analyses. We separately assembled the genomes of two representative spores using DNA sequences of 19 and 22 individually amplified nuclei. The genomes were compared with previously published data from other members of Glomeraceae including two strains of F. mosseae. No significant differences were observed among the species in terms of gene content, while the single nucleotide polymorphism density was higher in the strains of F. geosporum than in the strains of F. mosseae. In this study, we demonstrate that it is possible to sequence and assemble genomes from AM fungal spores sampled in the field, which opens up the possibility to include uncultured AM fungi in phylogenomic and comparative genomic analysis and to study genomic variation in natural populations of these important plant symbionts.

An Improved Technique for Isolation and Characterization of Single-Spore Isolates of Plasmodiophora brassicae.

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease that occurs in cruciferous crops worldwide. P. brassicae usually exists as a mixture of several pathotypes, which has hampered the research on resistance mechanisms of cruciferous crops against P. brassicae. In this study, clubroot galls were collected from a field in Shenyang, China, as a pathogen source to develop an efficient protocol for a single-spore isolation system of P. brassicae by optimizing the seedling age for inoculation, host inoculation method, and plant culture method. The operational steps of the single-spore isolation method were optimized as follows: the use of 2-day-old seedlings for inoculation, substituting a cryobox (100 × 2.0-ml vials) for culture dishes, the addition of nutrient solution culture, and microscopic observations of single spores. The rate of infection success was substantially improved, and single-spore isolates of four pathotypes (4, 8, 9, and 11) were acquired in this system. Subsequently, the optimized system was used to isolate and characterize the pathotypes of single-spore isolates of P. brassicae collected from five fields in regions in China. Approximately four to nine pathotypes were isolated from each region. Among these, pathotype 4 was the most prevalent. This study provides a source of valuable information that can eventually be used for the genetic analysis of host-P. brassicae interaction.

Variation in allele frequencies at the bg112 locus reveals unequal inheritance of nuclei in a dikaryotic isolate of the fungus Rhizophagus irregularis.

The genetic state of the arbuscular mycorrhizal fungus species Rhizophagus irregularis differs among isolates, including both homokaryotic and dikaryotic isolates. Via the production of multi-nucleate axexual spores, siblings of dikaryotic isolates may inherit unequal frequencies of nucleotypes. Using bg112, a microsatellite marker, previous studies revealed that lines deriving from single spores of the dikaryotic R. irregularis isolate C3 differed in their proportions of different alleles. A genomic study of single nuclei of R. irregularis, however, suggested that this marker was a multi-copy locus and that therefore it was inappropriate to study the inheritance of nuclei in dikaryotic isolates. In this study, we first analysed whole genome data of several R. irregularis isolates and demonstrated that bg112 is indeed a single copy locus in these genomes. Thus, the bg112 locus is a suitable marker to study the relative frequency of nucleotypes in R. irregularis. Second, by using amplicon sequencing, we confirmed the existence of one allele of bg112 in two homokaryotic isolates (DAOM197198 and C2) and two alleles in the dikaryotic isolate (C3). Finally, we found that the relative proportions of two bg112 alleles differed significantly among dikaryotic single-spore lines derived from isolate C3, indicating that genetically different nucleotypes are inherited unequally in this dikaryotic R. irregularis isolate.

Time-course of germination, initiation of mycelium proliferation and probability of visible growth and detectable AFB1 production of an isolate of Aspergillus flavus on pistachio extract agar.

The aim of this work was to assess the temporal relationship among quantified germination, mycelial growth and aflatoxin B1 (AFB1) production from colonies coming from single spores, in order to find the best way to predict as accurately as possible the presence of AFB1 at the early stages of contamination. Germination, mycelial growth, probability of growth and probability of AFB1 production of an isolate of Aspergillus flavus were determined at 25 °C and two water activities (0.85 and 0.87) on 3% Pistachio Extract Agar (PEA). The percentage of germinated spores versus time was fitted to the modified Gompertz equation for the estimation of the germination parameters (geometrical germination time and germination rate). The radial growth curve for each colony was fitted to a linear model for the estimation of the apparent lag time for growth and the growth rate, and besides the time to visible growth was estimated. Binary data obtained from growth and AFB1 studies were modeled using logistic regression analysis. Both water activities led to a similar fungal growth and AFB1 production. In this study, given the suboptimal set conditions, it has been observed that germination is a stage far from the AFB1 production process. Once the probability of growth started to increase it took 6 days to produce AFB1, and when probability of growth was 100%, only a 40-57% probability of detection of AFB1 production was predicted. Moreover, colony sizes with a radius of 1-2 mm could be a helpful indicator of the possible AFB1 contamination in the commodity. Despite growth models may overestimate the presence of AFB1, their use would be a helpful tool for producers and manufacturers; from our data 5% probability of AFB1 production (initiation of production) would occur when a minimum of 60% probability of growth is observed. Legal restrictions are quite severe for these toxins, thus their control from the early stages of contamination throughout the food chain is of paramount importance.

Characterization of arbuscular mycorrhizal fungal species associating with Zea mays.

Taxonomic identification of arbuscular mycorrhizal (AM) fungal spores extracted directly from the field is sometimes difficult because spores are often degraded or parasitized by other organisms. Single-spore inoculation of a suitable host plant allows for establishing monosporic cultures of AM fungi. This study aimed to propagate AM fungal spores isolated from maize soil using single spores for morphological characterization. First, trap cultures were established to trigger the sporulation of AM fungal species. Second, trap cultures were established with individual morphotypes by picking up only one spore under a dissecting microscope and transferring it to a small triangle of sterilized filter paper, which was then carefully inoculated below a root from germinated sorghum seeds in each pot and covered with a sterile substrate. All pots were placed in sunbags and maintained in a plant growth room for 120 days. Spores obtained from single spore trap cultures from each treatment, maize after oats (MO), maize after maize (MM), maize after peas (MP), and maize after soybean (MS), were extracted using the sieving method. Healthy spores were selected for morphological analysis. Direct PCR was conducted by crushing spores in RNAlater and applying three sets of primer pairs: ITS1 × ITS4, NS31 × AML2, and SSUmcf and LSUmBr. Nucleotide sequences obtained from Sanger sequencing were aligned on MEGA X. The phylogenetic tree showed that the closest neighbors of the propagated AM fungal species belonged to the genera Claroideoglomus, Funneliformis, Gigaspora, Paraglomus, and Rhizophagus. The morphological characteristics were compared to the descriptive features of described species posted on the INVAM website, and they included Acaulospora cavernata, Diversispora spurca, Funneliformis geosporus, Funneliformis mosseae, Gigaspora clarus, Gigaspora margarita, Glomus macrosporum, Paraglomus occultum, and Rhizophagus intraradices. These findings can provide a great contribution to crop productivity and sustainable management of the agricultural ecosystem. Also, the isolate analyzed could be grouped into efficient promoters of growth and mycorrhization of maize independent of their geographical location.

A Basic Guide to the Propagation and Manipulation of the Clubroot Pathogen, Plasmodiophora brassicae.

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio-economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagation Support Protocol 1: Evans blue staining to identify resting spore viability Support Protocol 2: Storage of Plasmodiophora brassicae Basic Protocol 2: Generation of single spore isolates from P. brassicae field isolates Basic Protocol 3: Phenotyping of Plasmodiophora brassicae isolates Basic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting spores Basic Protocol 5: Molecular detection of Plasmodiophora brassicae.

Detection of rare variants among nuclei populating the arbuscular mycorrhizal fungal model species Rhizophagus irregularis DAOM197198.

Identifying genuine polymorphic variants is a significant challenge in sequence data analysis, although detecting low-frequency variants in sequence data is essential for estimating demographic parameters and investigating genetic processes, such as selection, within populations. Arbuscular mycorrhizal (AM) fungi are multinucleate organisms, in which individual nuclei collectively operate as a population, and the extent of genetic variation across nuclei has long been an area of scientific interest. In this study, we investigated the patterns of polymorphism discovery and the alternate allele frequency distribution by comparing polymorphism discovery in 2 distinct genomic sequence datasets of the AM fungus model species, Rhizophagus irregularis strain DAOM197198. The 2 datasets used in this study are publicly available and were generated either from pooled spores and hyphae or amplified single nuclei from a single spore. We also estimated the intraorganismal variation within the DAOM197198 strain. Our results showed that the 2 datasets exhibited different frequency patterns for discovered variants. The whole-organism dataset showed a distribution spanning low-, intermediate-, and high-frequency variants, whereas the single-nucleus dataset predominantly featured low-frequency variants with smaller proportions in intermediate and high frequencies. Furthermore, single nucleotide polymorphism density estimates within both the whole organism and individual nuclei confirmed the low intraorganismal variation of the DAOM197198 strain and that most variants are rare. Our study highlights the methodological challenges associated with detecting low-frequency variants in AM fungal whole-genome sequence data and demonstrates that alternate alleles can be reliably identified in single nuclei of AM fungi.

An Overview of Mycoviral Curing Strategies Used in Evaluating Fungal Host Fitness.

The number of novel mycoviruses is increasing at a high pace due to advancements in sequencing technologies. As a result, an uncountable number of mycoviral sequences are available in public sequence repositories. However, only genomic information is not sufficient to understand the impact of mycoviruses on their host biology. Biological characterization is required to determine the nature of mycoviruses (cryptic, hypervirulent, or hypovirulent) and to search for mycoviruses with biocontrol and therapeutic potential. Currently, no particular selective method is used as the gold standard against these mycoviral infections. Given the importance of curing, we present an overview of procedures used in preparation of isogenic lines, along with their benefits and drawbacks. We concluded that a combination of single-spore isolation and hyphal tipping is the best fit for preparation of isogenic lines. Furthermore, recent bioinformatic approaches should be introduced in the field of mycovirology to predict virus-specific antivirals to get robust results.

Whole lifecycle observation of single-spore germinated Streptomyces using a nanogap-stabilized microfluidic chip.

Streptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular-level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of the entire lifecycle of Streptomyces development from the single-cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water-air interface. We used this chip to observe the lifecycle development of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron-scale water-air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A-Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single-spore heterogeneity, and secondary metabolism of single-spore germinated Streptomyces.

Single-Spore Extraction for Genetic Analyses of Arbuscular Mycorrhizal Fungi.

Biomass of arbuscular mycorrhizal fungi (AMF, Glomeromycota) is often only available in small quantities as these fungi are obligate biotrophs and many species are difficult to cultivate under controlled conditions. Here, I describe a simple, efficient approach to produce crude extracts from single or a small number of spores that can be used for genotyping AMF.

Quantifying the effect of water activity and storage temperature on single spore lag times of three moulds isolated from spoiled bakery products.

The inhibitory effect of water activity (aw) and storage temperature on single spore lag times of Aspergillus niger, Eurotium repens (Aspergillus pseudoglaucus) and Penicillium corylophilum strains isolated from spoiled bakery products, was quantified. A full factorial design was set up for each strain. Data were collected at levels of aw varying from 0.80 to 0.98 and temperature from 15 to 35°C. Experiments were performed on malt agar, at pH5.5. When growth was observed, ca 20 individual growth kinetics per condition were recorded up to 35days. Radius of the colony vs time was then fitted with the Buchanan primary model. For each experimental condition, a lag time variability was observed, it was characterized by its mean, standard deviation (sd) and 5th percentile, after a Normal distribution fit. As the environmental conditions became stressful (e.g. storage temperature and aw lower), mean and sd of single spore lag time distribution increased, indicating longer lag times and higher variability. The relationship between mean and sd followed a monotonous but not linear pattern, identical whatever the species. Next, secondary models were deployed to estimate the cardinal values (minimal, optimal and maximal temperatures, minimal water activity where no growth is observed anymore) for the three species. That enabled to confirm the observation made based on raw data analysis: concerning the temperature effect, A. niger behaviour was significantly different from E. repens and P. corylophilum: Topt of 37.4°C (standard deviation 1.4°C) instead of 27.1°C (1.4°C) and 25.2°C (1.2°C), respectively. Concerning the aw effect, from the three mould species, E. repens was the species able to grow at the lowest aw (awmin estimated to 0.74 (0.02)). Finally, results obtained with single spores were compared to findings from a previous study carried out at the population level (Dagnas et al., 2014). For short lag times (≤5days), there was no difference between lag time of the population (ca 2000 spores inoculated in one spot) and mean (nor 5th percentile) of single spore lag time distribution. In contrast, when lag time was longer, i.e. under more stressful conditions, there was a discrepancy between individual and population lag times (population lag times shorter than 5th percentiles of single spore lag time distribution), confirming a stochastic process. Finally, the temperature cardinal values estimated with single spores were found to be similar to those obtained at the population level, whatever the species. All these findings will be used to describe better mould spore lag time variability and then to predict more accurately bakery product shelf-life.

Comments on "Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China. Toxins 2015, 7, 3858-3875"- in Attributing Ochratoxin A Biosynthesis Within the Genus Penicillium Occurring on Natural Agricultural Produce.

The unusual attribution of trace amounts of ochratoxin A in some Chinese food commodities to Penicillium polonicum is questioned by European experience in searches for ochratoxinogenic food-spoilage Penicillia, where mistaken attribution is now known to have been due to cryptic Penicillium verrucosum contamination. Consequently, selection of single-spore isolates is recommended as pre-requisite for attributing mycotoxin biosynthetic potential to fungi.

Assessment of relevant fungal species in clinical solid wastes.

The study aimed to determine the fungal diversity in clinical waste samples from a healthcare facility in Penang Malaysia. Different fungi species were detected in 83.75 % of the 92 clinical waste samples that were screened from different sections of the healthcare facility. One hundred fifty fungal isolates comprising of 8 genera and 36 species were obtained. They were purified by using single spore isolation technique. Subsequently, the isolates were identified by phenotypic method based on morphological and culture characteristics on different culture media. Among all fungal isolates, Aspergillus spp. in section Nigri 10.2 %, Aspergillus niger 9.5 %, Aspergillus fumigatus 8.8 %, Penicillium. simplicissium 8 %, Aspergillus tubingensis 7.3 %, Aspergillus terreus var. terreus 6.6 %, Penicillium waksmanii 5.9 % and Curvularia lunata 6.5 % were the most frequent. Among five sections of the Wellness Centre, the clinical wastes collected from the diagnostic labs of haematology section had the highest numbers of fungal species (29 species). Glove wastes had the highest numbers of fungal species (19 species) among 17 types of clinical wastes screened. Among all fungal species, Aspergillus spp. exhibited higher growth at 37 °C than at 28 °C, indicating the potential of these opportunistic fungi to cause diseases in human. These results indicated the potential of hospital wastes as reservoirs for fungal species.

Modeling red cabbage seed extract effect on Penicillium corylophilum: Relationship between germination time, individual and population lag time.

The inhibitory effect of a red cabbage seed extract on germination time, individual (single spore) and population lag time of Penicillium corylophilum was studied. First, to compare the biological variability of single spore germination and lag times under stressful conditions, data were collected at levels of red cabbage seed extract varying from 0 to 10 mg/g (150 spores observed in each trial of germination, ca 50 spores in each individual lag experiment). Experiments were performed on malt agar at 25 °C, pH 5.2, aw 0.99. The data, without any transformation, were statistically analyzed; several probability distribution functions were used to fit the cumulated germination times and the individual lag times of spores. In both cases, the best fit was obtained with the Normal distribution. In parallel, lag times at the population level (ca 2000 spores per trial) were collected for the same range of plant extract. Not surprisingly, the difference between individual and population lag times could be explained by a stochastic process. More interestingly, it was shown that under stressful conditions, the population lag time did not correspond to the time required for germination of 95% of spores, but to a much longer time. Finally, it was deduced from the statistical analysis, completed by microscopic observations, that the plant extract affected mainly the hyphal elongation (and then the lag time) and not the germination. Next, secondary models were developed to quantify the effect of red cabbage seed extract on the median of germination times, individual and population lag times. The Minimum Inhibitory Concentrations (MICs) were estimated. It was shown that the red cabbage seed extract MIC for P. corylophilum lag time did not depend on the inoculum load. Application of the secondary models allowed us to conclude that under the conditions of our experiment, the addition of 10 mg/g of red cabbage seed extract enabled extension of lag time to two weeks.

Evaluation of Volvariella volvacea Strains for Yield and Diseases/Insect-Pests Resistance Using Composted Substrate of Paddy Straw and Cotton Mill Wastes.

Out of the 3 parent strains and 4 single spore isolates of Volvariella volvacea evaluated, strain, OE-274 gave earliest yield in 11.25-11.50 days post-spawning in all 4 trials. The yield varied in different strains in different trials and it was highest in strain, OE-272 in trial 1, SSI, OE-55-08 in trial 2, and strain, OE-274 in trial 3 and 4. In overall average, highest yield was in strain, OE-272, closely followed by strain, OE-274. The number of fruiting bodies per q substrate also varied in different strains in different trials. Highest numbers were in strain, OE-272, SSIs, OE-55-08 and OE-12-22, and strain, OE-210 in trial 1, 2, 3 and 4, respectively. Highest fruiting body wt was in strain, OE-274 in all 4 trials. The yield during different weeks of cropping varied in different strains but invariably it was highest in first week, which accounted for 60-70% of the total yield. The fruiting bodies of strain, OE-274 were of bigger size, brownish, toughest and with least tendency of veil opening, while that of strain, OE-272 and SSI, OE-55-08 were whitish to grayish-white, oblong, medium size, delicate and lesser tendency of veil opening. The strain, OE-274 and SSI, OE-55-08 exhibited higher resistance against the growth of competitor moulds and infestations of insect-pests, while strain, OE-272 exhibited highest susceptibility to insect-pests infestation.

Chinese Cabbage Clubroot Pathogen, Plasmodiophora brassicae, Is Genetically Stable.

Single spore isolates of Plasmodiophora brassicae e4 and e9 obtained from diseased Chinese cabbage were identified as race 4 and race 9, respectively, by the Williams' differential variety set. To confirm the possibility of variation in same generation and progeny of a single spore isolate of P. brassicae, random amplified polymorphic DNA (RAPD) analysis was conducted using the URP 3, 6 and OPA 7 primers. There was no difference in band type at each part of the gall of Chinese cabbage obtained by inoculation of e4 and e9 and amplification using the URP 3 and 6 primers when the same generation was analyzed. In addition, the progeny analysis, which was expanded to the third generation and conducted using the URP 3 and OPA 7 primers, revealed no differences in the band type of the e4 isolate. Based on these results, the single spore isolate of P. brassicae was genetically stable.