Dynamic utilization of low-molecular-weight organic substrates across a microbial growth rate gradient

Constantly in flux, low-molecular-weight organic substances (LMWOSs) are at the nexus between microorganisms, plant roots, detritus, and the soil mineral matrix. Nominal oxidation state of carbon (NOSC) has been put forward as one way to parameterize microbial uptake rates of LMWOSs and efficiency of carbon incorporation into new biomass. In this study, we employed an ecophysiological approach to test these proposed relationships using targeted exometabolomics (1H-NMR, HR-LCMS) coupled with stable isotope (13C) probing. We assessed the role of compound class and oxidation state on uptake kinetics and substrate-specific carbon use efficiency (SUE) during the growth of three model soil microorganisms (Penicillium spinulosum, Paraburkholderia solitsugae, and Ralstonia pickettii) in media containing 34 common LMWOSs. Microbial isolates were chosen to span a gradient in growth rate (0.046-0.316 hr−1) and differ phylogenetically (a fungal isolate and two bacterial isolates). Clustered, co-utilization of LMWOSs occured for all three organisms, but temporal cluster separation was most apparent for P. solitsugae. Potential trends (p <0.05) for early utilization of more oxidized substrates were present for the two bacterial isolates (P. solitsugae and R. pickettii), but high variability (R2 > 0.15) and a small effect of NOSC indicate these are not useful relationships for prediction. The SUEs ranged from 0.16-0.99 and the hypothesized inverse relationship between NOSC and SUE was not observed. Thus, our results do not provide compelling support for NOSC as a predictive tool, implying that metabolic strategies of organisms may be more important than chemical identity in determining LMWOS cycling in soils.ImportanceCommunity-level observations from soils indicate that low-molecular-weight compounds of higher oxidation state tend to be depleted from soil solution faster and incorporated less efficiently into microbial biomass under oxic conditions. Here, we tested hypothetical relationships between substrate chemical characteristics and the order of substrate utilization by aerobic heterotrophs at the population-level in culture, using two bacterial isolates (Ralstonia pickettii and Paraburkholderia solitsugae) and one fungal isolate from soil (Penicillium spinulosum). We found weak relationships indicating earlier uptake of more oxidized substrates by the two bacterial isolates but no relationship for the fungal isolate. We found no relationship between substrate identity and substrate use efficiency. Our findings indicate that substrate chemical characteristics have limited utility for modeling the depletion of low-molecular-weight organics from soil solution and incorporation into biomass over broader phylogenetic gradients.

A new record of psychrotrophic Paecilomyces formosus (Eurotiales: Ascomycota) from India: morphological and molecular characterization

A filamentous fungus Paecilomyces formosus (Eurotiales, Ascomycota) was detected for the first time from the region while surveying fungal diversity of a cold arid high-altitude pass (4,000 msl) located in Kargil district (Ladakh), India. The fungal isolate was characterized morphologically with camera lucida drawings and microphotographs, and identified using internal transcribed spacer (ITS) ribosomal DNA sequences. P. formosus has not been reported from India, or from arid/semi-arid/cold regions before, thus this represents a new record of Indian hot/cold desert mycoflora that is psychrotrophic in contrast to the more common thermophilic fungi.

HaplotypeTools: a toolkit for accurately identifying recombination and recombinant genotypes

Background Identifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis. Results HaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from https://github.com/rhysf/HaplotypeTools under the MIT License. Conclusions HaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.

Fungi Causing Post-Harvest Spoilage Carica papaya Linn Fruits of Two Selected Markets in Kano State, Nigeria

This study was carried out for four month (September, 2015 to December, 2015) to find the fungal species associated with post-harvest spoilage of pawpaw fruits sales at Na’ibawa and Wudil Markets all in Kano State. A total of five strains of fungi were isolated and identified as Aspergillus niger, Aspergillus flavuus, Rhizophus stolonifer, Yeast, and Fusarium. Pathogenicity test of each fungal isolate showed that all fungi were capable of causing rotting of the fruit and most severe rotting was caused by Aspergillus niger and Yeast with 8(29.63%) Respectively at Na’ibawa market and 10(34.48%) and 9(31.03%) at Wudil market followed by Rhizophus with 7(25.93%) and 5 (17.25%) while Aspergillus flavus and Fusarium produced the least rotting of the fruit. The difference in the fungal spoilage of Carica papaya between the two market areas Na’ibawa and Wudil markets was statistically significant (p≥0.05). From the result of this study, it is clearly indicated that the spoilage of pawpaw fruits in the two markets was due to fungal attack.

First report of leaf spot disease caused by Stemphylium eturmiunum on garlic in Korea.

Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 – 22 × 16 – 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch’s postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in Korea.

Characterization and Cellulase Production Activity of different Aspergillus Sp. Isolated from “Puri, Odisha” Marine Fungal Strains

Cellulose undergoes hydrolysis utilizing chemicals to deliver glucose, which might be utilized for the production of ethanol, organic acids, and various chemicals. Cellulases are a collection of hydrolytic catalysts that can hydrolyze the most plentiful natural polymer for example cellulose into smaller sugar components including glucose subunits. Cellulase is overpriced and contributes simply half to the general expense of hydrolysis because of the low explicit activity. This enzyme has enormous potential in industries and its use in food, beverages, textile, laundry, paper, and mash industries, and so forth. Consequently, there has been a lot of examination focused on new microorganisms producing cellulose enzymes with higher specific activities and greater efficiency. Currently, work is pointed toward screening and disconnecting cellulolytic growths from the marine samples gathered from the Bay of Bengal, Puri coast, Odisha. All out 7 fungi were isolated from these dirt examples, out of which 2 fungi were portraying the extensive cellulase activity. The fungal isolate, for example, MWF-1 andMSF-6 isolated from water silt respectively were recognized to show the most extreme zone of hydrolysis of carboxy-methyl cellulose. The cellulase activity was assayed by Carboxymethylcellulose "CMCase" (endoglucanase) measure.

First report of Arcopilus aureus causing leaf black spot disease of Pseudostellaria heterophylla in China

Pseudostellaria heterophylla (family Caryophyllaceae) is a perennial herbaceous plant. Its tuberous roots are highly valued in traditional Chinese medicine. It is mainly cultivated in a geo-authentic production zone located in the Guizhou, Anhui, Shandong, and Fujian provinces of China (Zhao et al. 2016). The herb is widely used for treating lung diseases and as a spleen tonic (Pang et al. 2011). A severe leaf black spot disease was observed on P. heterophylla in China, from 2018 to 2020. Plants displayed water-soaking symptoms in the early stage of infection, then the watery areas turned brown-red and a black mold appeared on the lesions. At a later stage, the leaf spots showed concentric rings surrounded by a yellow halo, and the initial infection site became dry and necrotic (Supplementary Figure S1). Nine infected plants were collected from three cultivation fields in Shibing County (N 27°4'21", E 108°8'0"), Guizhou province, on April 13th, 2019. The fungus was consistently isolated from symptomatic leaves on potato dextrose agar (PDA) medium according to the method described by Larran et al (2002). A total of 22 isolates were obtained, including 7 isolates of Arcopilus and 15 isolates of Trichoderma. The growth rates of isolate MJ2-2b on PDA and oatmeal agar (OA) medium were 3 to 5 mm/day at 25 °C (Supplementary Figure S2A and S2B). Mycelium of isolate MJ2-2b was dense, yellowish-brown on PDA, while it was sparse, bright-red on OA. Also, the mycelium secreted brownish-red pigment on both PDA and OA. Ascomata when mature were water drop and limoniform. Lateral hairs were brown, erect or flexuous, tapering towards the tips. Ascospores when mature were greyish-white to grey, limoniform, or fusiform to pyriform (Supplementary Figure S2C and S2D). Further, the beta-tubulin gene (Tub2) of the fungus was amplified by using primer pairs T1 and TUB4Rd as described by Wang et al (2016) and subjected to sequencing. NCBI nucleotide BLAST results showed that sequences from seven isolates had a 99.86% identity with A. aureus (strain ChL-C, GenBank accession No. MG889987.1) (Supplementary Figure S2F). Molecular phylogenetic analysis by maximum likelihood method using MEGA 7 confirmed that the fungal isolate clustered with A. aureus. Hence, the causal agent was identified as A. aureus based on morphological and molecular characteristics. The sequence was deposited in GenBank (accession No. MW531453). Pathogenicity tests were conducted on 15-day old tissue-cultured seedlings according to Ghanbary et al (2018) (Supplementary Figure S3). Leaves of 16 seedlings were inoculated with 1×1 mm 5-day-old PDA-grown mycelial plugsof the fungal isolate. The experiment was repeated 3 times. After 10 days, the inoculated leaves showed the same symptoms observed on plants in the field. The associated fungal pathogen was consistently re-isolated from the inoculated seedlings and identified by Tub2 gene sequencing. At present, there are no reports of A. aureus causing disease of plants. To the best of our knowledge, this is the first report of leaf black spot disease on P. heterophylla caused by A. aureus in China.

ISOLATION AND MORPHOLOGICALLY IDENTIFICATION OF ASPERGILLUS FLAVUS INCIDENCES FROM MAIZE SEEDS IN ABUJA, NIGERIA

Food safety and security well implemented could help in making more crops available for consumption. Maize seed is a crop well known to be attacked by fungi such as Aspergillus flavus and reduce its nutrients. This study intends to isolate and morphologically identify the Aspergillus flavus from maize seeds from Abuja, Nigeria. The experimental design was complete randomized design involving untreated yellow (Y) and white (W) maize seeds from7 locations in Abuja, Nigeria. Pure culture of fungal isolate was prepared using Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). Isolates obtained were characterized and identified on the basis of their colonial and morphological characteristics which include macroscopic and microscopic examinations. All maize seeds from the Abaji had no fungi incidences in both SDA and PDA, while all maize seeds from the experimental field show fungi incidences in both SDA and PDA. On SDA, the maize color yellow and white had F (2, 6) static values of 7.083 and 0.212 at p=0.129 and 0.941 respectively. For the PDA, white maize seeds and yellow maize seeds from all the locations had F (2, 6) static values of 0.377 and 0.521 at p=0.850 and 0.773. Aspergillus flavus was isolated in this study. The maize seeds from Kuje district and the experimental field 2 show a high incidence records. All growth of the Aspergillus in the two media used were not significant at p>0.05.