ITS rDNA Sequence Analysis Research Articles

Comprehensive analysis of microbiome biodiversity in popular date palm (Phoenix dactylifera L.) fruit varieties

Due to its nutritional value and health benefits, the date palm (Phoenix dactylifera L.) is an essential dietary food crop throughout Middle Eastern and African countries. Consumers are concerned about the possible microbial contamination of dates, especially since most dates arriving in local markets are unprocessed. The absence of processing increases the possibility of microbial contamination, which raises the probability of microbial contamination. This study aims to analyze and evaluate the variability of fungal and bacterial microbiota identified in the most popular date palm fruits in Saudi Arabia. The study assessed ten date variety fruits from the most popular date palm varieties for consumption in Saudi Arabia and analyzed the microbial count. Morphological and molecular characterization and comparison of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences identified 78 fungi, including 36 distinct species across 15 fungal genera. Alternaria, Fusarium, Curvilaria, Aspergillus, and Penicillium were the most frequent genera among the ten fruit cultivars studied, according to ITS-rDNA sequence analysis. Furthermore, 36 bacterial isolates were obtained from ten date varieties studied, each with a unique colony morphology. These isolates were identified based on sequence alignment and comparison of their 16S rDNA internal spacer regions to those available in public databases. The results showed that the bacterial isolates included 15 species from five bacterial genera. The results suggested that Bacillus, Stenotrophomonas, and Brucella were the prevailing genera among the ten tested fruit varieties. Some bacterial genera, such as Brucella, Achromobacter, and Stenotrophomonas, are well-known potential human pathogens. Chaetomium globosum was also recognized as air pollution causing adverse health effects such as allergies and as the causal agent of human fungal infections among the tested date varieties; the Rashodiah type exhibited the highest fungal contamination, whereas the Sagai variety displayed the lowest fungal contamination. Conversely, the Sukkari, Barhi, and Mejdool varieties were the most contaminated with bacteria among the ten tested varieties, while the Khalas variety showed the least bacterial contamination. To the best of the authors' knowledge, this study provides the initial comprehensive account of the molecular and morphological identification of all fungal and bacterial genera associated with date palm (P. dactylifera) fruits.

Diversity and Isolation of Endophytic Fungi in Panax japonicus and Biotransformation Activity on Saponins.

This study reports the diversity and community structure differences of the endophytic fungi of Panax japonicus of different ages to obtain novel endophytic fungi with glycoside hydrolytic activity for rare saponins production. This study used the high-throughput sequencing method to analyze the diversity and community structure of endophytic fungi of P. japonicus. The endophytic fungi were processed by traditional isolation, culture, conservation, and ITS rDNA sequence analyses. Then the total saponins of P. japonicus were used as the substrate to evaluate the glycoside hydrolytic activity. The composition analysis of the community structure showed that the abundance, evenness, and diversity of endophytic fungi of nine-year-old P. japonicus were the best among all samples. A total of 210 endophytic fungi were isolated from P. japonicus samples and further annotated by sequencing the internal transcribed spacer. Then the biotransformation activity of obtained strains was further examined on total saponins of P. japonicus (TSPJ), with a strain identified as Fusarium equiseti (No.30) from 7-year-old P. japonicus showing significant glycoside hydrolytic activity on TSPJ, including ginsenoside Ro→zinglbroside R1, pseudoginsenoside RT1→pseudoginsenoside RP1, chikusetsusaponin IV→tarasaponin VI and chikusetsusaponin IVa →calenduloside E. These results reveal the diversity and community structure differences of the endophytic fungi of P. japonicus with different ages and establish a resource library of endophytic fungi of P. japonicus. More importantly, we identified a valuable endophytic fungus with glycoside hydrolytic activity and provided a promising convenient microbial transformation approach to produce minor deglycosylated ginsenosides.

Biocontrol potential of an artificial synthetic bacterial consortium against peony root rot disease

Tree peony (Paeonia suffruticosa), a traditional Chinese cultivated flowering shrub and a prominent landscaping plant, is subject to root rot. The key symptoms of root rot include black roots and the yellowing of leaves. Biological control is an effective and eco-friendly strategy for managing this disease. This study focused on the isolation, identification, and biological control of the pathogens causing peony root rot. Utilizing tissue isolation, pathogenicity determination, and rDNA-ITS sequence analysis, Fusarium solani and Fusarium oxysporum were determined to be the primary causal agents of peony root rot. Illumina MiSeq high-throughput sequencing of 16 s-rDNA was used to investigate and characterize bacterial community structure and diversity in healthy and diseased peony roots, rhizosphere, and bulk soil. Results indicated that the presence of pathogenic fungi influences the structure of the rhizosphere bacterial community and that peony roots exhibit a strong selective effect on root bacterial colonization. Variations in the composition of the endophytic microbial community in healthy and diseased roots exceeded the variation in the rhizosphere. Proteobacteria and Actinobacteria are the dominant taxa in the rhizosphere and among root endophytes, comprising 90–96 % of the bacterial microbiota. The rhizosphere of healthy plants exhibits a significant enrichment in Proteobacteria (79.6 %) and Actinobacteria (14.9 %), while root endophytes in healthy plats exhibit enrichment in Proteobacteria (83.5 %). In contrast, the rhizosphere and root endophytes in diseased plants are abundant in Proteobacteria (69 % and 66.1 %, respectively). Notably, nine strains of biocontrol bacteria were isolated. Three synthetic bacterial consortia were then constructed based on inhibitory assays and the dissolving rates of phosphorus and potassium exhibited by the candidate bacteria. The synthetic bacterial consortia were evaluated for biocontrol and growth promotion properties. The constructed synthetic consortium with the best performance reduced the average morbidity and mortality of treated plants by 27.59 % and 55.56 %, respectively, compared to the untreated control. In summary, synthetic bacterial consortium I (SCI) exhibited the best disease control and growth-promoting effects on tree peony. Using a synthetic bacterial consortium represents a new and novel approach to the biological control of peony root rot.

Phylogeny and Fungal Community Structures of Helotiales Associated with Sclerotial Disease of Mulberry Fruits in China.

Mulberry fruit sclerotiniose is a prevalent disease caused by the fungal species Ciboria shiraiana, C. carunculoides, and Scleromitrula shiraiana of the order Helotiales, and severely affects the production of mulberry. However, these species have only been identified using morphological and rDNA-ITS sequence analyses, and their genetic variation is unclear. To address this, morphological and two-locus (ITS and RPB2) phylogenetic analyses were conducted using culture-dependent and independent methods for 49 samples from 31 orchards across four provinces in China. Illumina MiSeq sequencing was used to assess the fungal communities obtained from fruits varying in disease severity and color from an orchard in Wuhan. Conidial suspensions of C. shiraiana and C. carunculoides isolated from diseased fruits, diseased fruits affected with hypertrophy and pellet sorosis sclerotiniose, and mycelia of Sclerotinia sclerotiorum were determined to be pathogenic to the mulberry cultivar YSD10. However, fruits inoculated with S. sclerotiorum mycelia exhibited nontypical disease symptoms, and mycelia and conidia obtained from C. carunculoides and S. shiraiana strains were not pathogenic. Maximum parsimony and Bayesian analyses using the sequences of the assessed loci indicated species variability with no evidence of geographic specialization. Metagenomic analysis revealed that the diversity of fungal communities was reduced with disease progression. Furthermore, within a single fruit, the presence of two Ciboria spp. was detected. These results provide novel insights into Ciboria spp., revealing the secondary infections caused by conidia in diseased fruits, genetic variations of the pathogens, and the occurrence of coinfection. This improved understanding of fungal pathogens will aid in developing effective disease control strategies.

β-glycosidase activity associated with the formation of aroma compounds in native non-Saccharomyces yeasts isolated from cocoa bean fermentation

Description of the subject. The use of non-Saccharomyces yeast for improving fine cocoa flavor perception has been poorly explored. Objectives. In this study, we investigated the contribution of the non-Saccharomyces yeasts producing β-glycosidase isolated from the spontaneous fermentation of cocoa to the quality and production of aromas during the alcoholic fermentation of a medium based on cocoa pulp. Method. The screening for β-glycosidase activity and molecular identification of non-Saccharomyces yeast isolates from Amazonian cocoa bean fermentations were performed on agar plates with esculin as a substrate and by 5.8S ITS rDNA sequence analysis, respectively. The yeasts producing β-glycosidases were used as a starter culture for alcoholic micro-fermentations of a medium based on cocoa pulp, incubated at 30 °C for 48 h. The quantification of organic acids, ethanol, and sugars was obtained by HPLC, and aroma compounds after cocoa pulp fermentation were identified by GC-FID and GC-MS. Results. Twenty-six non-Saccharomyces yeast isolates were selected by β-glycosidase activity, comprising species such as Pichia kudriavzevii (n = 15), Pichia sp. (n = 2), Pichia sporocuriosa (n = 1), Candida orthopsilosis (n = 3), Issatchenkia sp. (n = 1), I. orientalis (n = 1), and P. kudriavzevii/I. orientalis (n = 3). All yeast strains exhibited rapid growth in cocoa pulp medium, low ethanol production, high organic acid production, and higher glucose consume than fructose. These yeasts produced thirty different volatile compounds. The main groups included alcohols (3), esters (19), terpenes (4), phenols (2), organic acids (2), and aldehydes (3), with 67% of these having fruity and floral aromas. Pichia sp., P. kudriavzevii/I. orientalis and Issatchenckia sp. were the largest producers of volatile compounds. Notably, this study identified volatile compounds with fruity aromas previously unassociated with cocoa fermentations for the first time. Conclusions. These data demonstrate the potential of β-glycosidase-producing non-Saccharomyces yeasts to improve floral and fruity aromas, suggesting their promising use as a starter culture for cocoa fermentation.

Serendipita officinale sp. nov. (Serendipitaceae): a new species of orchid mycorrhizal fungus

Serendipita is a prominent fungal genus associated with orchids. In this study, a new species of orchid mycorrhizal fungus, Serendipita officinale, is described and thoroughly studied using morphological and molecular approaches. The fungus was isolated from naturally occurring protocorms of Dendrobium officinale in Chongqing Province, China. S. officinale has been observed to actively promote the germination of seeds and facilitate the seedling development of D. officinale, forming typical mycorrhizal pelotons within protocorm cells. This species is characterized by its characteristics on PDA medium, including the presence of abundant villiform and felty aerial mycelium, uninucleate hyphal cells, and the highest growth rate in terms of colony diameter (78 mm diam. in 2 weeks). Furthermore, analyses of ITS rDNA and LSU sequences also provide additional support for the novelty of this new plant symbiont.

Three New Species of Gongronella (Cunninghamellaceae, Mucorales) from Soil in Hainan, China Based on Morphology and Molecular Phylogeny.

The genus Gongronella is important in agriculture and industry by secreting various natural bioactive metabolites such as chitosanases and organic acids. During the most recent 8 years, a total of 14 new species have been described, remarkably enriching the diversity of this genus. In this study, we added three more new species to this valuable genus, based on a combination of morphological traits and phylogenetic information. Six strains of the genus Gongronella were isolated from soil collected in Hainan Province, China. Phylogenetic analyses of ITS and LSU rDNA sequences grouped these strains into three independent clades. According to their unique morphological characteristics, they were classified as G. multiramosa sp. nov., G. qichaensis sp. nov. and G. oleae sp. nov. The G. multiramosa was characterized by multiple branched sporangiophores and was closely related to G. pedratalhadensis. The G. qichaensis was characterized by obscure collars and closely related to G. butleri, G. hydei and G. banzhaoae. The G. oleae was characterized by the presence of oil droplets in the sporangiospores and was closely related to G. chlamydospora and G. multispora. Their descriptions and illustrations were provided, and their differences from morphological allies and phylogenetic-related species are discussed.

First Report of Acrophialophora jodhpurensis Causing Root Rot and Seedling Damping-Off of Pepper (Capsicum annum L.) in Karbala Province, Iraq

Pepper is an important vegetable crop in Iraq and worldwide. During a survey conducted in 2022 in Karbala Province, Iraq, an epidemic root rot disease affecting pepper plants was observed. Based on morphological appearances, pathogenicity assessment and rDNA-ITS sequence analyses, the fungal causing this disease was identified as Acrophialophora jodhpurensis (OQ102241.1). To best of our knowledge, this is the first record of A. jodhpurensis fungus causing root rot on pepper crop in Karbala, Iraq.

A new Report of Trichocladium griseum in Iraq: Morphological and Molecular Descriptions

During a survey of diversity of fungal pathogens in nurseries of Babylon and Karbala Provinces of Iraq in 2021. Trichocladium griseum associated with diseased ornamental plants, Gazania sp. and Carpobrotus edulis was isolated and identified relied on its morphological features, ITS rDNA sequencing and phylogenetic analysis. The T. griseum fungus has not yet been described previously in Iraq. Hence, this is the first report of Trichocladium griseum presence in Iraq.

Underreported and taxonomically problematic: characterization of sanguinicolid larvae from freshwater limpets (Burnupiidae), with comments on the phylogeny and intermediate hosts of sanguinicolids.

Blood flukes of freshwater fish are understudied worldwide. Consequently, genetic information and data on their intramolluscan stages are scarce. In the current study, freshwater limpets of the genus Burnupia (Burnupiidae) from South Africa were examined for digeneans. Of 1645 specimens, 3.10% were infected by Sanguinicolidae larvae. Four sanguinicolids were distinguished by body size, number of penetration glands, tegumental spines’ patterns and relative sizes of the finfolds on the body and furcae. Analyses of 28S, 18S and ITS rDNA sequences showed that the morphotypes were distinct from each other and from sanguinicolids whose genetic data are available. The present study is the first genetic characterization of sanguinicolids from Africa. Phylogenetic reconstruction revealed that the present species clustered with a sanguinicolid from Poland and were sister to Sanguinicola and Pseudosanguinicola from Russia and USA, respectively. The results indicate that the current species represent an unknown genus. What is more, blood fluke sequences from East Africa (presumed to be sanguinicolids), were distant from Sanguinicolidae and showed a closer relationship with acipensericolids from the USA. Freshwater fish blood flukes seem to be more diverse than previously recorded and use species of at least 13 gastropod families as intermediate hosts.

Xylaria sridharii sp. nov.—a new species of Xylariaceae from India

A new species of Xylaria associated with leaflets of coconut (Cocos nucifera) in Konaje, Mangalore, Karnataka is collected and examined. The species was characterized by having long stromata terminated with a globose head-like swollen fertile part, which showed a further extended thread-like appendage at its tip. Micro-morphological examination, cultural characterization and molecular sequence analysis of ITS-rDNA, LSU and TUB were carried out. The phylogenetic analysis based on combined ITS-rDNA and tub2 gene sequences showed that Xylaria sridharii is phylogenetically related to X. oxyacanthae, X. reevesiae and X. palmicola. However, X. sridharii is distinguished by the presence of the hirsute stromata, which shows multiple branching (2–4, but rarely recorded up to 6) at the apex, and each branch produces a fertile part, which in turn ends with a thin filiform appendage.

First Report of Cladosporium tenuissimum causing Postharvest rot of Elaeagnus conferta Fruit in Kunming, China.

Elaeagnus conferta Roxb. is a perennial evergreen climbing shrub and is mainly native to India, Vietnam, Malaysia, and South China (Gupta & Singh, 2021). Various parts of this plant are used to treat multiple diseases(Gupta et al., 2021). Between during the months of March and April of 2021, in Kunming city of grower fields, Yunnan Province (N 25°02'; E 102°42'), southwest China. Some postharvest E. conferta fruits showed brown spots of decay with a greyish mycelium, which symptom only appears on fruit, and did not find it on this plant. The incidence of this disease in postharvest E. conferta fruits ranges from 45 % to 65 % in natural conditions. This pathogen is harmful and causes many plant diseases. Such as rice, oriental persimmon, pear, panicles of mango, and so on (Cho & Shin, 2004; Guillén-Sánchez et al., 2007; Lee et al., 2009). The infected fruit samples surface was disinfected with 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, then aseptic water washing three times. The fruit tissue is rich in carbohydrates and water content, which aid the growth of fungal species. Putting these diseased tissues on a potato dextrose agar (PDA) medium, cultured at 25 ± 1 ℃ for 7 days. The colonies grow on the PDA medium, then separated and puried again. Three pure cultures (YNGH01, YNGH03, YNGH05) were obtained, which were stored in 15 % glycerol at -80 ℃ refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Agricultural University. After 7 days of cultivation, the colonies were round and the diameter attained up to 38 mm, the surface of the colony showed tiled, fluffy, with a velvet-like texture, greyish-green to smoke-gray, slightly raised in the middle, the edges were radial hollow and wrinkle (Fig. 1A). Conidiophores were solitary, erect, unbranched or rarely branched, slightly flexuous at the apex, septate, dark brown, 254 to 680 µm long, 3.6 to 4.5 µm wide, top of the conidiophores or the rostral were slightly swollen (Fig. 1B). Conidia were light gray or grey, solitary or bispora, irregular in shape and size (Fig. 1C), nearly circular (3.21 × 3.31 µm), oval to lemon-shaped (6.59 × 3.21 µm) or elliptical (8.35 × 4.65 µm). The CTAB method extracts 3 isolates (YNGH01, YNGH03, YNGH05) genomic DNA (Aboul-Maaty & Oraby, 2019). To confirm identity with molecular identification, performed by three different genomic DNA regions, fragments of internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) genomic regions. These genomic DNA were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Carbone & Kohn, 1999). The sequences of these isolates were uploaded to GenBank (YNGH01: ON753810, ON868696, ON912090 YNGH03: ON753812, ON868698, ON912092, and YNGH05: ON753814, ON868700, ON912094). NCBI's BLASTn search of those ITS sequences showed 99.81% similar to C. tenuissimum (MG873077.1), and sequences TEF-1α and ACT were 100% identical to several isolates of C. tenuissimum (OM256526.1 and MT154171.1). Combined the ITS region, TEF-1α, and actin (ACT) genomic regions of isolates YNGH01, YNGH03 and YNGH05 to construct a phylogenetic tree with MEGA11. Maximum likelihood phylogenetic analyses further confirmed the results (Fig. 2)(Santos et al., 2020). Healthy and mature E. conferta fruits were used for pathogenicity test. Pathogens were washed with sterilized water at a final concentration of 2× 106 spores/mL (Jo et al., 2018). The test was divided into A and B groups (A: The surface of fruits was pierced with a sterilized needle that carried pathogenic fungus of final concentration at 2×106 spores/mL B: Sprayed at the concentration of 2×106 spores/mL on fruits). The control fruits were treated with sterilized water and stored at 25 ± 1 ℃ with a relative humidity of 80 %, average group with 10 fruits in this test, which was repeated three times. After 7 days, the fruits of group A were initially sesame seed size of the disease spots, nearly round, irregular, with grayish-brown spots, and slightly depressed. Later, the lesion gradually turns dark brown (Fig. 1D). And group B began with small patches of brown fungal growth on the pericarp, with the development of the disease, the necrotic spots enlarged and developed irregular and coalesced, the color of spots became gray or black gradually (Fig. 1E). The symptoms were similar to previously observed and the pathogen was reisolated and identified as C. tenuissimum. Control fruits were healthy (Fig. 1F). The pathogens test fulfilled Koch's postulates. According to morphology (Bensch et al., 2012), rDNA-ITS, TEF-1α, and ACT sequence analysis, phylogenetic analysis, and pathogenicity test, the pathogen was identified as C. tenuissimum. To our knowledge, this is the first report of C. tenuissimum occurring on E. conferta fruits in China.

Determination of polysaccharide and phenolic contents and antioxidant activity of the crude extract from mycelial biomass of strain Tropicoporus linteus NTH-PL3

Tropicoporus (synonym Phellinus) mushroom is one of the precious medicinal mushrooms and has been proven to play a significant role in supporting health. However, studies on the Tropicoporus mushroom have only been carried out in our country within the last ten years, so the scientific data on this mushroom source is not really rich. This study aimed to add research data on molecular identification of strain NTH-PL3 collected in nature and determination of bioactive compounds, as well as antioxidant activity of the total extract from mycelium of Tropicoporus. Results of rDNA ITS sequence analysis showed that strain NTH-PL3 belongs to Tropicoporus linteus species with the highest similarity in ITS sequence of 99.87%. The total yield of bioactive compounds extracted from the mycelium using 75% methanol was 29.75±3.84%. In the crude bioactive extract, total polysaccharide content was 23.72±1.34 mg glucose/g and 18.54±1.87 mg gallic acid/g for phenolic. EC50 values for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and ferrous ion chelating ability of total crude bioactive extract from mycelium of Tropicoporuswere 268.54±3.89 and 411.65±10.87 μg/ml, respectively.

First Report of Mucor inaequisporus Dade (Mucorales, Mucoromycota) causing Postharvest rot of Strawberry Fruit in Kunming, China.

Strawberry (Fragaria × ananassa Duch.) is an important and very popular fruit around the world because they have unique taste, special fragrance and rich nutrition (Hashmi et al., 2013; Neri et al., 2015). However, mature fruits have the characteristics of smooth texture, high softening rate and re-breathing rate were easily infected by various pathogens (Lara et al., 2004). In June 2022, a few postharvest strawberry fruits were found to rot, soften and cover with a green or yellow mycelium in Kunming city, Yunnan Province (25°02' N; 102°42' E), southwest China. The symptoms of fruits initially observed a tiny white mycelium and water damage, then the mycelium became yellow, with water-soaked areas extended rapidly. Eventually, the entire fruit was covered with yellow, dense mycelium (Fig. 1A, B). The incidence of this postharvest decay in strawberry fruits ranged from 35 to 45% and caused serious loss. This pathogen was isolated from diseased fruit tissues, transferred to potato dextrose agar (PDA) and malt extract agar (MEA), and incubated in the dark at 25 ± 1 ℃. Then purify and separate again. Four pure strains (BDFM1 to BDMF4) were obtained and stored in 15% glycerol at -80 ℃ in the State Key Laboratory for Conservation and Utilization of Bio-Resources Room 1012, Yunnan Agricultural University. On PDA, the colonies were initially white and the reverse was light yellow, after 5 days, the hyphae became golden yellow, and the diameter of the colony reached 5 cm (Fig. 1C). On MEA, the colonies were initially light yellow, and after 4 days, yellow-orange and the reverse were yellow to dark yellow (Fig. 1D). Unbranched sporangiophores are up to 26.7-30.8 μm diameter with columellae frequently pyriform, oblong or ellipsoid, obovoid (43- 60 × 90-135 μm), colorless or yellowish (Fig. 1E), simple or with long or short sympodial branches. Sporangia yellow, globose or subglobose, 55.3-123.7 μm in diameter, wall echinulate, which are spherical formed at the top of the main sporangiophore and each branch (Fig. 1F), Sporangiospores yellow, which are spherical (5.0-8.5 × 5.5-9 μm), oval (21.5-7 × 11-20 μm), thin and smooth-walled. To further identify the fungus, the total genomic DNA was extracted from four strains using the CATB method (Aboul-Maaty & Oraby, 2019). The partial fragments of internal transcribed spacer (ITS), and partial 28S rRNA gene sequence were amplified with the primers ITS1/ITS4, and LR0R/LR5 respectively. These sequences of strains BDFM1 to BDMF4 were uploaded to NCBI GenBank as accessions ON951610, ON951611, ON951612 and ON951613, OP430532, OP430533 OP430534 and OP430535. Using NCBI's BLASTn tools, the nucleotide sequences of strains showed 96.12 - 99.21 % identity to JN206177 (M. inaequisporus strain CBS 496.66). BDFM1 to BDMF4 isolates were used to MEGA11 construct a phylogenetic tree. Maximum likelihood phylogenetic analyses (Fig. 2) and phylogenetic tree from Bayesian method (Fig. 3) further confirmed the results. Pathogenicity tests were conducted with healthy strawberry fruits. We selected two stains (BDFM1, BDFM2) from four same isolates for this test, cultured on MEA for 4 days, then washed with sterilized water and the spore suspensions were adjusted at 1.0 × 106 spores/ml. Ten fruits were surface-disinfected with 75% alcohol for 15 s, rinsed with sterile water 3 times, then air-dried, and sprayed with the spore suspension on the surface, while the control fruits were sprayed with sterile water. The fruits were incubated at 26 °C with 90% relative humidity in a plastic container. This test was performed in triplicate. After 2 days, the fruits sprayed with the spore suspensions showed light yellow hyphae (Fig. 1H), which covered one-third of the fruit surface (Fig. 1G). After 4 days, yellow-orange mycelia covered the entire fruit, with yellow oil-droplets at the tip of the mycelium (Fig. I). Control fruits remained healthy. The fungus was re-isolated and identified as Mucor inaequisporus thus completing Koch's postulates. Mucor inaequisporus was identified by symptoms, morphology (de Freitas et al., 2021), rDNA-ITS sequence analysis, and pathogenicity test. As we know, this is the first report of M. inaequisporus on strawberry fruits in China.

Screening Trichoderma strains isolated from soil growing Curcuma longa in Hung Yen province for treating waste after turmeric processing

The cultivation and processing of turmeric are developing, resulting in a large amount of turmeric waste causing serious environmental pollution. Trichodermafungi are potential microbial sources for agricultural waste treatment thanks to their ability to synthesise several extracellular enzymes. In this study, 15 strains of Trichoderma were isolated from turmeric-growing soil in Hung Yen province. Among them, 4 strains, which were named HYT11, HYT12, HYT13, and HYT14, were found to have a high potential for synthesising cellulase, amylase, and protease enzymes. Based on morphological characteristics and ITS rDNA sequence analysis, 3 strains (HYT11, HYT12, and HYT13) were identified as Trichoderma asperellum and the strain HYT14 was identified as Trichoderma harzianum. All four strains showed the ability to grow and form conidia on turmeric waste. The study also determined the optimal conditions for the conidia formation of these strains, including a nutrient medium containing potato extract (200 g/l) supplemented with 2% glucose, 1% peptone, and 2% agar, and the cultivation of 7 days at 30oC. In summary, the research has selected four Trichodermastrains, HYT11, HYT12, HYT13, and HYT14, which are promising for application in treating turmeric waste.

Low genetic heterogeneity of Leishmania major in different geographical regions of Iran.

To examine the genetic diversity of Leishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019-2021. The Leishmania ITS-rDNA gene was amplified and Leishmania sp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity of L. major in human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity of L. major was observed in Southwest/Southeast Iran (Hd: 0.104-0.286). The statistically Fst value indicated that L. major is not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.

Characterization of cereal cyst nematodes in wheat using morphometrics, SCAR markers, RFLP, and rDNA-ITS sequence analyses

Characterization of cereal cyst nematodes in wheat using morphometrics, SCAR markers, RFLP, and rDNA-ITS sequence analyses

Endophytic Fungi Isolated from Ageratina adenophora Exhibits Potential Antimicrobial Activity against Multidrug-Resistant Staphylococcus aureus.

Multidrug-resistant bacteria such as Staphylococcus aureus (MRSA) cause infections that are difficult to treat globally, even with current available antibiotics. Therefore, there is an urgent need to search for novel antibiotics to tackle this problem. Endophytes are a potential source of novel bioactive compounds; however, the harnessing of novel pharmacological compounds from endophytes is infinite. Therefore, this study was designed to identify endophytic fungi (from Ageratina adenophora) with antibacterial activity against multidrug-resistant bacteria. Using fungal morphology and ITS-rDNA, endophytic fungi with antibacterial activities were isolated from A. adenophora. The results of the ITS rDNA sequence analysis showed that a total of 124 morphotype strains were identified. In addition, Species richness (S, 52), Margalef index (D/, 7.3337), Shannon-Wiener index (H/,3.6745), and Simpson's diversity index (D, 0.9304) showed that A. adenophora have abundant endophytic fungi resources. Furthermore, the results of the agar well diffusion showed that the Penicillium sclerotigenum, Diaporthe kochmanii, and Pestalotiopsis trachycarpicola endophytic fungi's ethyl acetate extracts showed moderate antibacterial and bactericidal activities, against methicillin-resistant Staphylococcus aureus (MRSA) SMU3194, with a MIC of 0.5-1 mg/mL and a MBC of 1-2 mg/mL. In summary, A. adenophora contains endophytic fungi resources that can be pharmacologically utilized, especially as antibacterial drugs.

Indigenous Yeasts from Rose Oil Distillation Wastewater and Their Capacity for Biotransformation of Phenolics

The indigenous yeasts associated with the spontaneous fermentation of phenolic-rich rose oil distillation wastewater (RODW) generated after the industrial distillation of rose oil were studied. The ITS-rDNA sequence analysis of the samples collected from RODW fermented at semi-sterile conditions, a waste deposition lagoon and endophytic yeasts isolated from industrially cultivated Rosa damascena suggests that the spontaneous RODW fermentation is caused by yeasts from the genus Cyberlindnera found also as endophytes in the rose flowers. Phylogenetic analysis based on the nucleotide sequences of the translation elongation factor (TEF1α) and 18S- and 26S- rRNA genes further confirmed the taxonomic affiliation of the RODW yeast isolates with the genus Cyberlindnera. The RODW fermentation capacity of a selected set of indigenous yeast isolates was studied and compared with those of common yeast strains. The indigenous yeast isolates demonstrated a superior growth rate, resulting in a nearly double reduction in the phenolic content in the fermented RODW. The indigenous yeasts' fermentation changed the RODW phenolics' composition. The levels of some particular phenolic glycosides decreased through the depletion and fermentation of their sugar moiety. Hence, the relative abundance of the corresponding aglycons and other phenolic compounds increased. The capacity for the biotransformation of RODW phenolics by indigenous yeasts is discussed.

MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF AN EDIBLE RUSSULA MUSHROOM IN NORTHEAST VIETNAM

Wild mushrooms are extensively gathered by rural communities throughout the uplands of northern Vietnam and nearby countries for food and medicine. These non-wood forest products are important for the livelihood of numerous rural communities. Many of these fungi form symbiotic relationships with the roots of certain species of forest trees, and the fruiting bodies can not be produced in artificial media. In the northeast region of the country, an edible red Russula (Nấm chẹo or Nấm hồng cô) has been harvested from under wild Castanopsis, Engelhardia and Lithocarpus trees by local people for a long time. However, the identity of this Russula is unknown. We collected basidiomes of this edible Russula in Cao Bang, Bac Giang and Quang Ninh provinces and described its morphological features. We used ITS rDNA and LSU rDNA sequence analysis to compare with other similar taxa in GenBank. Morphological and molecular analysis identified the taxon as Russula griseocarnosa, previously described from southern Yunnan, China. There is strong interest by local governments to commercialize R. griseocarnosa in Vietnam. However, over-harvesting could have detrimental effects on the population of this edible species. Therefore, further studies are needed to quantify the ecological requirements and to develop sustainable harvesting protocols of this edible species in Vietnam.