Internal Transcribed Spacer Region Research Articles

First Report of Phytophthora cinnamomi causing root rot of avocado trees in Greece.

In September 2023, thirty declining 30-year-old avocado (Persea americana) trees ('Hass' grafted on 'Zutano' seedlings) were detected in a 1.5-ha orchard in the island of Crete (Chania region). Crown symptoms encompassed wilting and leaf chlorosis, advancing to defoliation and extensive dieback. Tap and feeder roots decayed and brown discoloration of root tissues was evident on heavily infected trees. The disease was severe and widespread, resulting in a 5% mortality rate among 300 trees. The pathogen was isolated with a modified soil baiting technique (Ferguson and Jeffers, 1999). Surface disinfected avocado fruits were immersed in water containing soil samples. Following a period of 2 to 8 days, tissue fragments from the resulting necrotic lesions on the fruit surface were transferred on ΡΑRP medium and subsequently incubated at 20°C (Ferguson and Jeffers, 1999). Three isolates (AV2, AV12 and AV11a) were obtained by transferring single hyphal tips to new Petri dishes containing V8 juice agar. They were grown at 20˚C and used for identification after 10 days. Isolates formed coralloid colonies with abundant clustered spherical hyphal swellings and terminal or intercalary (ratio 1:5) thick-walled chlamydospores measuring 20 to 36 μm (avg 29±0.8 μm) with characteristic thick walls (avg 1.2±0.2 μm). Sporangia, produced in non-sterile soil-extract water, were ovoid to obpyriform, persistent, non-papillate, 32 to 81 μm (avg 56±4.8 μm) long and 20 to 42 μm (avg 31±3.2 μm) wide (n=100). Isolates were heterothallic as they did not produce oospores in single cultures. Based on the morphological traits the isolates were identified as Phytophthora cinnamomi (Erwin and Ribeiro 1996). The internal transcribe spacer region (ITS) including ITS1, 5.8S rDNA region, and ITS2 as well as the cytochrome c oxidase subunit I (coxI) gene of the three representative isolates wereamplified with ITS1/ITS4 and FM83/FM84 primers, respectively (White et al. 1990; Martin and Tooley 2003), and sequenced (GenBank acc. PP506613 to PP506615 and PQ063867 to PQ063869, respectively). BLAST search revealed almost 100% identity with the sequences of P. cinnamomi ex-isotype isolate (KC478663 and KU899315 respectively). Pathogenicity tests using isolate AV2 were conducted following the soil infestation method (Jung et al. 1996) using six-year-old avocado 'Zutano' seedlings. Six non-inoculated plants treated with vermiculite-multivitamin juice mixture were used as controls. Plants (1 m tall) were grown in pots under greenhouse conditions and watered regularly. Six weeks post inoculation, all inoculated trees showed chlorosis, wilting and root rot, while control plants remained symptomless. Symptoms were similar to those observed in the field and the pathogen was re-isolated and molecularly identified as previously described. This study presents the first documented occurrence of P. cinnamomi, widely regarded as the most destructive avocado pathogen globally, on avocado crops in Greece (Rodger et al. 2019). Additionally, this marks the first recorded presence of this pathogen on the island of Crete, regardless of the host species. The accurate identification of Phytophthora species associated with avocado root rot is essential for implementing an effective disease management strategy, particularly in the selection of appropriate disease-resistant rootstocks.

Full-length ITS amplicon sequencing resolves Phytophthora species in surface waters used for orchard irrigation in California's San Joaquin Valley.

Diverse Phytophthora species, including many important plant pathogens, have been widely detected among surface water irrigation sources. In the past decade, metabarcoding has been used to characterize waterborne Phytophthora populations. Metabarcoding typically involves amplification of portions of the nuclear ribosomal internal transcribed spacer (ITS)1 or ITS2 from Phytophthora species, followed by indexed high throughput sequencing. However, full-length sequences of the entire ITS region are required for resolution of many Phytophthora species. We used metabarcoding with PacBio sequencing of full-length ITS amplicons to analyze populations of Phytophthora in waterways of the Stockton East Water District (SEWD) in the northern San Joaquin Valley of California. This approach yielded species-level resolution of many members of the Phytophthora community. Results were compared to those obtained by using ITS1 or ITS2 regions alone and were found to provide superior species resolution for P. pini, P. capsici, and P. gregata. Samples were collected throughout the 2021 irrigation season from five waterways across the SEWD. Thirty-eight Phytophthora species were detected in the waterways, including tree-crop pathogens P. acerina, P. cactorum, P. pini, P. ×cambivora, P. niederhauserii, P. mediterranea, and P. taxon walnut. These pathogenic species were detected throughout the SEWD during most of the irrigation season. The results demonstrated the utility of full-length ITS amplicon sequencing for identifying Phytophthora species in environmental samples and suggested that some disease risk may be incurred by orchardists irrigating with SEWD water. Additional epidemiological studies will be required to critically evaluate this risk.

First Report of Root Rot on Rhizome of Polygonatum kingianum Caused by Aspergillus awamori

Polygonatum kingianum Coll. et Hemsl (Huangjing), which belongs to the family Asparagaceae, is a perennial traditional Chinese herb with homologous medicinal and edible value (Liu et al., 2021). Huangjing is known to promote blood circulation; it has anti-inflammatory properties, increases immunity, and provides hypoglycemic treatments (Ma et al., 2019). Root rot-infected P. kingianum exhibited withering yellow leaves and stems, rhizome rot, slowed growth, and plant death. In recent years, with an average incidence of up to 45%, the spread of HJ root rot (rhizome and stem bases) has resulted in a significant reduction in the quality and up to 63% reduction in the yields of Sichuan Junlian (104.5°E, 28.2°N) and Guizhou Zhunyi (107.0°E, 27.7°N). After collecting the diseased samples, we used the tissue isolation method to isolate the pathogenic fungi (Wu et al., 2020). Four fungal isolates associated with root rot were obtained: HJ-G2 (two strains), HJ-G3 (one strain), HJ-G4 (one strain), and HJ-G6 (two strains), of which HJ-G2 and HJ-G6 were the dominant species. To determine pathogenicity of each strain, tests were conducted by wounding rhizomes wth an inoculation needle and the pathogen strain was inoculated onto the wound and symptoms observed. The results reveal that HJ-G6 exhibited the strongest pathogenicity against P. kingianum (Figure 1). The HJ-G6 colonies were black, grew rapidly, and produced a large number of spores (Figure 1). A spherical apical sac (conidial head) is formed at the top with two palisades of cells, metulae and phialides, which are shaped radially and produce a large number of spores with 2-5 um in diameter (Figure 2). Morphological observations revealed that the isolate was consistent with Aspergillus awamori (Naher et al., 2021). To further confirm the fungal species, the ribosomal internal transcribed spacer (ITS), β-tubulin (TUB), and elongation factor 1-alpha (EF-1a) gene regions were amplified with ITS1/ITS4, Bt2a/ Bt2b, and EF1/EF2. Primer and PCR amplification were performed as previously described (Paul et al., 2017). The sequences were compared with those obtained from GenBank. The ITS sequences (GenBank accession number OR682143) of the isolates (HJ-G6) were 100% identical to those of the strain PANCOM10 (GenBank accession number MT007535.1) of Aspergillus awamori. The EF-1a sequences (GenBank accession OR752352) of the isolates (HJ-G6) were 98% identical with strain ITEM 4777 (GenBank accession FN665402.1) of Aspergillus awamori. The TUB sequences (GenBank accession number OR752351) of the isolate (HJ-G6) were 100% identical with strain AF158 (GenBank accession MH781275.1) of Aspergillus awamori. Three maximum likelihood trees were constructed using MEGA v5.0 (Kumar et al., 2018) based on the sequences (ITS, TUB, and EF-1a) of the HJ-G6 strain and that of Aspergillus spp. previously deposited in GenBank (Paul et al., 2017). Phylogenetic analysis showed that HJ-G6 belonged to the Aspergillus awamori clade (Figure 3). Combined with morphological analysis and DNA sequencing, HJ-G6 was identified as Aspergillus awamori. To verify pathogenicity, P. kingianum roots were inoculated with the colonized agar discs of the isolates. P. kingianum plants inoculated with uncolonized agar discs were used as controls. After inoculation, P. kingianum roots were moved to the inoculation chamber under high humidity at 28 °C for 1 d and then transferred to a greenhouse. Previous studies have reported that Fusarium sp. are root rot pathogens in the rhizomes of medicinal plants (Pang et al., 2022; Song et al., 2023). In this study, HJ-G2, HJ-G3, and HJ-G4 were used as the positive controls. Typical symptoms of root rot appeared 3 days after inoculation and were similar to those observed in the field, whereas the control plants remained symptomless. According to the results of the inoculation experiment, the pathogenicity of Aspergillus awamori to P. kingianum root rot was significantly stronger than that of Fusarium (Figure 1). The pathogen was isolated from the rotting root of P. kingianum and the ITS region was sequenced again. Alignment analysis of the ITS sequences revealed that the causal agents were consistent with those of the original isolates. These studies fulfill Koch’s postulates. As far as we know, this is the first report of Aspergillus awamori causing root rot in P. kingianum.

First Report of Neofusicoccum yunnanense Causing Leaf Spot on Ivy in Yunnan Province, China.

Ivy (Hedera nepalensis var. sinensis (Tobl.) Rehd) is an evergreen root-climbing vine, widely cultivated in eastern Asia because of its ornamental, environmental, and medicinal value (Wu et al. 2019). In October 2023, the leaf spot symptom of Ivy was observed in the Kunming Botanical Garden in Yunnan Province, China (25.14°N, 102.75°E), and the disease incidence was up to 38% (76 of 200 leaves). Initially, dark-brown or black small spots appeared on the leaves. As the lesions progressed, their center emerged tawny, and the black halos were expanded around the lesions. In severe cases, small spots could link together to form leaf blight even resulting in blade death. In order to obtain pure isolates, 10 diseased leaves were collected and cut into 1 mm × 1 mm pieces, surface disinfected with 75 % ethanol for 30 s, followed by 3% NaClO for 3 min, and finally washed three times with sterilized water. The pieces were placed on the potato dextrose agar (PDA) media, which was incubated at 25°C for 3 days. Individual hyphal tips from the developing fungal colonies were placed on PDA and incubated for 5 to 10 days. Six strains (6 out of 10) were obtained with same colony morphology. Conidia were hyaline, unicellular, nonseptate, ellipsoidal or fusiform, thin walled, externally smooth and ranged from 15.0 to 22.0 (avg. 18.4) μm × 5.0 to 8.0 (avg. 7.2) μm (n=30). Morphological comparison proposed that the present fungi belonged to Neofusicoccum (Zhang et al. 2021). Two isolates (GUCC23-0141 and GUCC23-0142) were selected for multi-gene phylogenetic analyses. The PCR reaction of the internal transcribed spacer region (ITS), translation elongation factor 1-α (EF1-α), and β-tubulin genes were run using primers ITS1/ ITS4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone and Kohn 1999), and Bt2a/ Bt2b (Glass and Donaldson 1995). The accession numbers of DNA sequences of GUCC23-0141 and GUCC23-0142 are ITS: PP728109 and PP728110; TUB2: PP744490 and PP744491; and TEF1-α: PP744488 and PP744489. BLAST analysis showed 100% identity for ITS and TUB2, and 98.97% for TEF1-α with the Neofusicoccum yunnanense (CSF6142). Phylogenetic analyses also supported that our isolates kept a close relationship to N. yunnanense. Three one-year plants were used for pathogenicity test, two of which were inoculated with PDA plugs containing N. yunnanense and one of which was inoculated with blank PDA plugs and used as control. For each plant, three leaves were selected to conduct the test, whose surfaces were sterilized with 75% alcohol. All the leaves were covered with cotton moistened with sterilized water on top. All plants were placed in a greenhouse with 25℃ and 75% humidity. Few small black spots were observed at the inoculation site after 3 days, which were enlarged gradually after 7 days. However, control plants remained healthy. N. yunnanense was reisolated from the diseased tissues and identified based on morphological and molecular characteristics. On basis of pathogen identification and Koch's test, we proposed the leaf spot of Ivy caused by N. yunnanense. This was the first report about N. yunnanense causing the disease of Ivy. This result provides a theoretical basis for further research into the control of the disease. As an important ornamental plant, we should pay attention to the management of this disease.

First report of anthracnose caused by Colletotrichum fructicola on winter jasmine (Jasminum nudiflorum) in China.

Winter jasmine (Jasminum nudiflorum Lindl.) is a medium-sized, deciduous shrub native to China that has become a popular choice among gardeners and landscapers. In 2020 to 2021, symptoms of anthracnose including brown necrotic spots, enlarged irregular lesions and leaf blight were observed on leaves of 20 winter jasmine shrubs in a public garden (22°34'58'' N; 113°56'23'' E) in Shenzhen, China, and with an estimated disease incidence of 65%. Tissues samples (6 × 6 mm2) surrounding the necrotic spots were surface sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 1 min, then rinsed with sterile water for three times and dried with sterile filter paper. Tissues were placed on potato dextrose agar (PDA) medium and incubated at 25℃. After 3 to 7 d, pure cultures were obtained by transferring hyphal tips to new plates and 32 isolates producing Colletotrichum-like colonies were obtained from 40 tissues (isolation frequency=32/(4×10)=80%). Three representative isolates YCH09, YCH23 and YCH32 were selected for further study. Three selected isolates were identical in morphological characteristics. Colonies on PDA after 5 d at 25℃ were white to gray with cottony mycelia and grayish-white on the underside of the culture. Conidia (n = 60) measured 15.4 ± 1.1 µm (13.0 to 17.1 µm) in length and 5.4 ± 0.3 µm (4.9 to 6.0 µm) in width and were hyaline, single-celled, cylindrical with rounded ends. Appressoria (n = 15) measured 7.1 ± 0.1 µm (5.3 to 8.9 µm) in length and 5.2 ± 0.2 µm (4.1 to 6.2 µm) in width and were brown to dark brown, ovoid. These morphological features were aligned with those of Colletotrichum spp. (Weir et al. 2012). Sequences of five genetic markers of representative isolates YCH09, YCH23 and YCH32 including the rDNA internal transcribed spacer region, chitin synthase, partial actin, β-tubulin 2 and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) region were 99.3 to 100% identical to the ex-type isolate of C. fructicola strain ICMP 18581 (Zhang et al., 2020). From the maximum likelihood phylogenetic tree which was constructed based on concatenated sequences, three representative isolates (YCH09, YCH23 and YCH32) were clustered with other isolates of C. fructicola. The above morphological and molecular characteristics suggest that causal agent was C. fructicola. Pathogenicity was tested using a whole-plant assay. Five healthy plants were inoculated by spraying a conidial suspension (1.5×104 conidia/ml; 20 ml per plant) of the isolate YCH23 onto the foliage (Marshall et al., 2023). Three noninoculated control plants were sprayed with sterile water. All plants were placed in a greenhouse at 25±2℃ with approximately 75% relative humidity. Yellow lesions appeared on leaves of inoculated plants as early as 4 days after inoculation (DAI), and irregularly shaped brown spots similar to those observed in the field were formed on 10 DAI. Noninoculated plants remained asymptomatic. Colletotrichum isolates resembling morphological characters of YCH23 were reisolated from all inoculated plants, then identified as C. fructicola by DNA sequence analysis. C. fructicola is a well-known fungus causing anthracnose on more than 63 plant species including agricultural and horticultural plants worldwide (Talhinhas and Baroncelli, 2021). To our knowledge, this is the first report of C. fructicola infecting J. nudiflorum plants in China. Since its potential risk to other horticultural plant species, precautions may be necessary to minimize the spread of this fungi.

Internal transcribed spacer as effective molecular marker for the detection of natural hybridization between the bivalves Pinna nobilis and Pinna rudis.

The Pinna nobilis, a Mediterranean mollusc, has suffered population declines due to a massive mortality event associated with various factors including the parasite Haplosporidium pinnae. Some populations show resilience, possibly due to local environmental conditions. In this study, a molecular multiplex PCR method was developed using species-specific primers targeting Internal Transcribed Spacer (ITS) regions of P. nobilis and P. rudis, allowing accurate species identification and hybrid detection. Samples from Mediterranean areas were analysed, including putative hybrids and individuals from five other bivalve species. DNA was isolated, ITS regions were amplified and sequenced, and phylogenetic analyses confirmed species differentiation and primer specificity. The multiplex-PCR successfully identified P. nobilis, P. rudis, and their hybrids based on distinct amplicon patterns. This study highlights the value of molecular tools in species conservation, especially for monitoring and managing hybridization, supporting effective biodiversity conservation strategies.

Inopinatus corneliae sp. nov. gen. nov. isolated from human skin: A newly discovered keratinophilic hyphomycete, order Onygenales.

Fungi clinically relevant to human skin comprise prevalent commensals and well-known pathogens. Only rarely human skin harbours fungi that evade identification. To characterise an enigmatic specimen isolated from a skin lesion. A comprehensive clinical and mycological workup including conventional methods for phenotypic characterisation and sequencing based on internal transcribed spacer (ITS) and large subunit (LSU) regions to infer a phylogenetic tree. Cultures on common solid media were macroscopically inconspicuous initially until mycelial tufts developed on the surface, notably on potato dextrose agar. Polymorphous chlamydospores were detected but no aleurospores and ascomata. At 26°C, the isolate grew on standard agars, plant materials and garden soil and utilised peptone, keratins, lipids, inulin, erythrocytes and cellulose. It also grew at 5°C and at 37°C. Nucleotide sequences of its ITS region showed 93% similarity to sequences of different Malbranchea species. The closest matches among LSU rRNA sequences were obtained with the genera Amauroascus, Arthroderma, Auxarthronopsis and Malbranchea (93%-95%). A combined phylogenetic analysis placed the fungus in a sister clade to Neogymnomycetaceae, classified as incertae sedis in Onygenales, on a large distance to either Diploospora rosea or 'Amauroascus' aureus. The genus Inopinatus gen. nov. (MB854685) with the species Inopinatus corneliae sp. nov. (MB854687) is introduced to accommodate our isolate (holotype: DSM 116806; isotypes: CBS 151104, IHEM 29063). Probably Inopinatus corneliae is a geophilic species that, although potentially harmful, was no relevant pathogen in our case. Its ecology, epidemiology and pathogenicity need to be further clarified.

Cyberlindnera qingyuanensis f.a., sp. nov., a yeast species isolated from rotting wood.

Two yeast strains were isolated from rotting wood samples collected on Qingyuan Mountain, Fujian Province, PR China. Phylogenetic analysis, based on the concatenated sequences of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit rRNA gene, revealed that these two strains represent a novel species of the genus Cyberlindnera. The proposed name for this new species is Cyberlindnera qingyuanensis f.a., sp. nov. (holotype: GDMCC 2.300; ex-type: PYCC 9925) although the formation of ascospores was not observed. The novel species differs from its close relative Cyberlindnera galapagoensis by 7.7% sequence divergence (37 substitutions and seven indels) in the D1/D2 domain and 9.7% sequence divergence (42 substitutions and 34 indels) in the ITS region, respectively. Additionally, Cyb. qingyuanensis differs from its close relative Cyb. galapagoensis by its ability to grow in cellobiose, l-rhamnose, ribitol, galactitol, and dl-lactate, its growth at 37 °C, and its inability to ferment raffinose. The Mycobank number is MB 854693.

Additional new species of Xenodidymella from pasture-medicinal plants in Iran.

Xenodidymella species have a wide range of hosts and can be found as pathogens and saprobes. In this study, two new species of Xenodidymella were found from leaf diseases of three pasture-medicinal plants in Ilam Province, in the west of Iran, and proposed here as X. ilamica and X. scandicis spp. nov. These species were identified based on morphological features and phylogenetic analyses of the internal transcribed spacer regions 1 & 2 and 5.8S nrDNA (ITS), partial beta-tubulin gene (tub2), and partial RNA polymerase II second largest subunit (rpb2) gene. The four Xenodidymella strains isolated in this study were delimited into two sister clades, with the two isolates of X. ilamica from the leaf spot of Colchicum speciosum and Ficaria kochii and two isolates of X. scandicis from leaf blight of Scandix pecten-veneris. Morphologically, X. scandicis produces larger, ostiolate or poroid pycnidia in vitro, while pycnidia in the cultures of X. ilamica are non-ostiolate and smaller. Some pycnidia in old cultures of X. scandicis produce a neck, but a distinct neck in X. ilamica has not been observed. Moreover, three plants under study are new hosts for the genus Xenodidymella.

Prolonged treatment of dermatophytosis caused by Trichophyton indotinea with terbinafine or itraconazole impacts better outcomes irrespective of mutation in the squalene epoxidase gene.

Over the past decades, the increasing incidence of recurrent dermatophytosis associated with terbinafine-resistant Trichophyton has posed a serious challenge in management of dermatophytosis. Independent reports of failure of treatment and high minimum inhibitory concentrations (MIC) of antifungals are available, but data correlating MIC and clinical outcomes is still sparse. Therefore, the present study was conducted to evaluate the outcomes of systemic treatment of dermatophytosis and its correlation with MIC of the etiological agents isolated from such patients. Retrospective analysis of 587 consecutive patients with dermatophytosis was done from March 2017 to March 2019. Demographic and clinical details of the patients were noted, along with the results of direct microscopy and fungal culture. The isolates were identified by sequencing the internal transcribed spacer region of rDNA. Antifungal susceptibility testing was performed following the CLSI M38 protocol. Mutation in the squalene epoxidase (SE) gene was detected by DNA sequencing and ARMS-PCR. Based on the culture-positivity and prescribed systemic antifungal, patients were categorised into Group I culture-positive cases treated with systemic terbinafine and Group II culture-positive cases treated with systemic itraconazole, each for a total period of 12 weeks. In the present study, 477 (81.39%) were culture-positive; however, 12 weeks follow-up was available for 294 patients (Group I-157 and Group II-137) who were included for statistical analysis. In both groups [Group I-37/63 (51.4%) and Group II-14/54 (58.3%)], a better cure rate was observed if the initiation of therapy was performed within <6 months of illness. Treatment outcome revealed that if therapy was extended for 8-12 weeks, the odds of cure rate are significantly better (p < .001) with either itraconazole (Odd Ratio-15.5) or terbinafine (Odd Ratio-4.34). Higher MICs for terbinafine were noted in 41 cases (cured-18 and uncured-23) in Group I and 39 cases (cured-16 and uncured-23) in Group II. From cured (Group I-17/18; 94.4% and Group II-14/16; 87.5%) and uncured (Group I-20/23; 86.9% and Group II-21/23; 91.3%) cases had F397L mutation in the SE gene. No significant difference in cure rate was observed in patients with Trichophyton spp. having terbinafine MIC ≥ 1or <1 μg/mL (Group I-p = .712 and Group II-p = .69). This study revealed that prolonging terbinafine or itraconazole therapy for beyond 8 weeks rather than the standard 4 weeks significantly increases the cure rate. Moreover, no correlation has been observed between antifungal susceptibility and clinical outcomes. The MIC remains the primary parameter for defining antifungal activity and predicting the potency of antifungal agents against specific fungi. However, predicting therapeutic success based solely on the MIC of a fungal strain is not always reliable, as studies have shown a poor correlation between invitro data and invivo outcomes. To address this issue, further correlation of antifungal susceptibility testing (AFST) data with clinical outcomes and therapeutic drug monitoring is needed. It also highlights that initiation of the treatment within <6 months of illness increases cure rates and reduces recurrence. Extensive research is warranted to establish a better treatment regime for dermatophytosis.

Tuber pathogenicity of Macrophomina phaseolina strain 3a isolated from rotten cassava tuber from farm lands in Osogbo, Osun State, Nigeria, its virulence genes and ADMET properties.

Macrophomina phaseolina is a pathogen that causes an opportunistic disease that spreads by soil and seeds and affects more than 500 different plant species, like fruits, trees, and row crops. Mycotoxins, such as phaseolinic acid, and phaseolinone, are produced by M. phaseolina isolates in previous investigations; however, the production of these mycotoxins seems to vary depending on the host and the region. In this study, Macrophomina phaseolina strain 3A was isolated from rotten cassava tuber and identified using the analysis of the sequences of the internal transcribed spacer region. The isolate was inoculated on a fresh healthy cassava tuber at 25°C and tuber-rotting potential was monitored for 4 weeks. Virulence genes MPH_06603, MPH_06955, and MPH_01521 were determined with designed primers, and secondary metabolites were characterized by FTIR and GCMS. The rotten tuber effect was observed from the 2nd week of the experiment with severe tuber rot and weight reduction. The PCR showed the presence of MPH_06603 virulence gene. The GCMS showed N-Methylpivalamide (115.0m/z), Butane, 1,4-dimethoxy- (119.0m/z), and 5-Hydroxymethylfurfural (126.0m/z) were the predominant metabolites produced by the pathogen. The compounds in the metabolites inhibit CYP3A4 enzymes, cause eye irritation, and Human Ether-a-go-go-related gene inhibition. This study revealed that M. phaseolina was responsible for the cassava tuber rot which leads to a lower yield of farm produce. The metabolites produced are toxic and unsafe for human consumption. It is suggested that farmers should destroy any cassava affected by this pathogen to prevent its toxic effects on humans and animals.

First DNA Barcoding Survey in Bulgaria Unveiled Huge Diversity of Yeasts in Insects.

In this study, we conducted a comprehensive survey aimed at assessing the diversity of yeast species inhabiting the guts of various insect species collected mainly from two Bulgarian National Parks, namely, Rila, and Pirin. The insect specimens encompass a broad taxonomic spectrum, including representatives from Coleoptera, Orthoptera, Lepidoptera, Hymenoptera, Dermaptera, Isopoda, and Collembola. Yeast strains were identified with DNA barcoding using the ribosomal markers, specifically, the D1/D2 domains of the ribosomal large subunit (LSU) and the internal transcribed spacers regions ITS 1 + 2 (ITS). The analysis unveiled the presence of 89 ascomycetous and 18 basidiomycetous yeast isolates associated with the insect specimens. Furthermore, our study identified 18 hitherto unknown yeast species.

Cercospora polygonatum, a New Species Causing Gray Leaf Spot Disease in Polygonatum cyrtonema.

This study identified a new species (Cercospora polygonatum) that causes gray leaf spot (GLS) disease in cultivated Polygonatum cyrtonema. This fungal species was isolated from the affected region of GLS on P. cyrtonema leaves. Pathogenicity bioassays were conducted based on Koch's postulates. Morphology was examined based on the features of conidiomata, conidiogenous loci, conidia/conidiophores, and conidiogenous cells. The rDNA internal transcribed spacer region, calmodulin, translation elongation factor 1-alpha, and histone genes were subjected to phylogenetic analysis using the MrBayes tool in Phylosuite. Bootstrap support analysis for phylogenetic placement confirmed the new species, which was significantly different from the closely related species C. senecionis-walkeri and C. zeae-maydis. The morphological characteristics also supported this finding, with the conidiogenous cells of C. polygonatum being considerably shorter than those of C. senecionis-walkeri or C. zeae-maydis. In addition, C. polygonatum was distinguished by its cultural characteristics. As this fungus was isolated from P. cyrtonema, it was named C. polygonatum F.Q. Yin, M. Liu & W.L. Ma, sp. nov. The type specimen (H8-2) was preserved at the China General Microbiological Culture Collection Center. This is the first report of GLS caused by C. polygonatum on P. cyrtonema leaves in China. The current study enriches the knowledge regarding Cercospora sp., contributes to the identification of a species causing GLS in P. cyrtonema, and provides useful information for the effective management of this disease.

Microsporum canis and Sporothrix schenckii: Fungi Causing Skin Infections in Cats

Companion animals such as cats help to reduce stress among people as they delight their owners in their ways. Good management and hygiene practices of pets help in keeping them in a healthy condition. Nevertheless, since fungal infection develops rapidly, there is a high tendency for them to get infected. The paucity of data regarding skin mycoses among cats in Malaysia leads to this study. Private veterinary clinics from the Central Region of Peninsular Malaysia were approached for participation in this study. Sampling was conducted for one year, collecting hair plucked, skin scrapings, and swabs from lesions of the cats with skin problems and inoculating onto Sabouraud Dextrose Agar media. Diagnosing the fungal colony was conducted through a direct examination method using lactophenol cotton blue stain and molecular identification of the isolates using polymerase chain reaction targeting the fungi species’ internal transcribed spacer region and β-tubulin gene. Of the 127 cats, 93 were positively infected, mainly with Microsporum canis (n = 38) and Sporothrix schenckii (n = 26). Saprophytic fungi detected on cats were Alternaria sp., Aspergillus sp., Candida sp., Chaetomium sp., Chrysosporium sp., Curvularia sp., Fusarium sp., Geotrichum sp., Penicillium sp., Talaromyces sp., Trichoderma sp., Trichosporon sp., and Xylaria sp. This finding represents the number of cats infected with fungal dermatitis in Selangor, Negeri Sembilan, the Federal Territory of Kuala Lumpur, and Putrajaya.

Fungicide resistance in Botrytis cinerea and identification of Botrytis species associated with blueberry in Michigan.

Botrytis blossom blight and fruit rot, caused by Botrytis cinerea, is a significant threat to blueberries, potentially resulting in substantial economic losses if not effectively managed. Despite the recommendation of various cultural and chemical practices to control this pathogen, there are widespread reports of fungicide resistance, leading to decreased efficacy. This study aimed to characterize the resistance profile of B. cinerea isolated from blighted blossoms and fruit in 2019, 2020 and 2022 (n = 131, 40, and 37 for the respective years). Eight fungicides (fludioxonil, thiabendazole, pyraclostrobin, boscalid, fluopyram, fenhexamid, iprodione, and cyprodinil) were tested using conidial germination at specific discriminatory doses. Additionally, 86 isolates were phylogenetically characterized using the internal transcribed spacer regions (ITS) and the protein coding genes: glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and RNA polymerase II second largest subunit (RPB2). This revealed higher fungicide resistance frequencies in 2020 and 2022 compared to 2019. Over all 3 years, over 80% of the isolates were sensitive to fludioxonil, fluopyram, and fenhexamid. Pyraclostrobin and boscalid showed the lowest sensitivity frequencies (<50%). While multi-fungicide resistance was observed in all the years, none of the isolates demonstrated simultaneous resistance to all tested fungicides. Botrytis cinerea was the most prevalent species among the isolates (74) with intraspecific diversity detected by the genes. Two isolates were found to be closely related to B. fabiopsis, B. galanthina, and B. caroliniana and 10 isolates appeared to be an undescribed species. This study reports the discovery of a potentially new species sympatric with B. cinerea on blueberries in Michigan.

First report of Phytopythium vexans causing gummosis and root rot of Khasi Mandarin (Citrus reticulata Blanco) in north eastern states of India.

Khasi mandarin (Citrus reticulata Blanco) is the most economically important crop among the citrus growing region in the north-eastern India (Singh et al. 2016). An extensive survey was conducted to identify the causal agent of citrus root rot and gummosis in north eastern states (Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam) of India during October 2021-23. The gummosis disease incidence ranged from 5 to 95 % in 10 to 25 years old Khasi mandarin plants showing relatively more chronic symptoms on mature trees. Yellowing and dropping of leaves, twigs die back, gum oozing from infected bark and loss of feeder roots were the typical symptoms of the disease. Infected bark tissue and young lemon leaf baits in rhizosphere soil were plated on corn meal agar medium supplemented with pimaricin (10 µg/ml), ampicillin (250 µg/ml), rifamycin (10 µg/ml) and 300µg/ml carbendazim and incubated at 26℃. Fifty isolates were purified and maintained on Carrot agar medium. These isolates showed similar cultural and morphological characteristics. Two representative isolates from Arunachal Pradesh (AP21 and AP26) were selected for further experiments and deposited to Indian Type culture collection (ITCC), New Delhi with accession no. 9156 and 9157 respectively. The colonies were fast growing, showing rosette pattern along with whitish blooming mycelium appearance with no visual sporulation at the surface. The hyphae were coenocytic with initially right-angled branching. Sporangia were globose or sub globose and papillated. Oogonia were smooth and globose (16.29-21.09 µm) in diameter. Antheridia were irregular, cylindrical and broadly attached to oogonia. Empty sporangia were also observed. Multilocus phylogenetic analysis using internal transcribed spacer region (Das et al. 2011), β tubulin (Blair et al. 2008) and Cytochrome oxidase II gene (Noireung et al. 2020) showed that these isolates formed a stable clade with Phytopythium vexans (CBS119.80) sequence retrieved from NCBI database. BLAST analysis showed that ITS sequence of AP21 (OQ372986) and AP26 (OQ381083) had >99 % identity with P. vexans isolate NS-3 (ON533631). Further, BLAST analysis of β tubulin (AP21 OQ446053, AP26 OR405377) and Cox II gene (AP21 OQ473414, AP26 OR552422) sequences showed that our Indian isolates showed >99 % similarity with P. vexans voucher strain CBS119.80. To fulfil Koch's postulates, Khasi mandarin (Citrus reticulata) seedlings were inoculated by adding 100 ml zoospore suspension of P. vexans (1x105 spores/ml) in sterilized soil (Thao et al. 2020). The experiment was carried out in triplicate. Yellowing of leaves and leaf drop were observed 7 days post inoculation while 30 days post inoculation, treated plants started showing symptoms of root rot, including mild root decay. No symptoms were observed in control treatment. The pathogen was reisolated from symptomatic roots and confirmed through colony and sporangium morphology. Recently, it was reported that P. vexans is associated with apple and pear decline in the Saiss plain of Morocco (Jabiri et al. 2021), root rot on mandarin in Thailand (Noireung et al. 2020) and on Durian in Vietnam (Thao et al. 2020). As per our knowledge, this is the first report of P. vexans causing root rot and gummosis in Khasi mandarin from north eastern states of India. This finding is significantly important for the development of a successful disease management strategy in India.

First Report of Leaf Blight of Osmanthus fragrans Caused by Diaporthe eres in China.

Osmanthus fragrans is an evergreen garden tree species, with high ecological, social, and economic benefits (Lan et al. 2023), which is widely planted in Guizhou Province. From late April to June 2023, a leaf blight disease was observed on O. fragrans in a bauxite mining area in Qingzhen City, with an incidence of ~50%. Symptoms first appeared at the leaf tip or margin, as irregular brown spots, which gradually coalesced into dark brown patches until the leaves withered and fell off. Symptomatic leaves were collected and surface disinfected with 2% NaClO for 30 s, 75% ethanol for 30 s, rinsed 3 times in sterile ddH2O, air-dried and placed on potato dextrose agar (PDA) medium and incubated at 25°C for 7 d. Fungal colonies on PDA of 9 similar obtained isolates were white, with at least one concentric ring. The reverse was light yellow and gradually turned brown. At 12 d, the pycnidia on PDA was gray to black, spherical or conical, with a diameter of 305.15 μm (n=20). The conidial horns oozed out from pycnidia after 25 d of incubation on Pinus massoniana needles. The alpha conidia were unicellular, fusiform, hyaline, had a guttule at each end, and measured 6.24 ± 0.10 μm × 2.48 ± 0.04 μm (n=50). No beta or gamma conidia were observed. The morphological characteristics were likely to Diaporthe spp. (Gomes et al. 2013). DNA of isolates GH02, GH06 and GH08 was extracted. The internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB2), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, CAL228F/CAL737R (Carbone and Kohn, 1999), βt2a/βt2b and CYLH3F/H3-1b (Crous et al. 2004; Glass and Donaldson, 1995), respectively. The sequences of ITS, TEF-1α, TUB2, CAL and HIS were deposited in GenBank (GH02: PP813499, PP813844, PP813846, PP813848 and PP813850; GH06: PP813500, PP813845, PP813847, PP813849 and PP813851; GH08: PP507168 and PP529956 to PP529959). BLAST results showed the sequences of GH08 were highly identical to sequences of Phomopsis mahothocarpi (NR147522 [ITS], 527/530), P. mahothocarpi (MW700277 [TEF-1α], 367/372), D. eres (OR885862 [TUB], 513/513), D. celeris (ON221721 [CAL], 484/486), and D. eres (OP968956 [HIS], 477/477). A phylogenetic tree constructed with MEGA X using Neighbor-Joining algorithm (Felsenstein, 1985) indicated the isolate GH02, GH06 and GH08 separated from D. eres CBS 297.77 previously reported from O. aquifolium in Netherlands, as well as D. osmanthi and D. fusicola from O. fragrans in China (Gomes et al. 2013; Long et al. 2019; Si et al. 2021). Based on these results, the three isolates were identified as D. eres (Chaisiri et al., 2021). The isolate GH08 was deposited in the Forest Protection Laboratory, Guizhou University. To confirm pathogenicity, spore suspensions (1×105 spores/mL) of GH08 were sprayed on healthy detached leaves (n=10) and leaves of 3-year-old potted O. fragrans seedlings (n=8). An equal volume of sterile water was sprayed for the control. Then they were placed at 20°C and 70-80% RH. Similar leaf blight symptoms appeared after 5 and 15d on the inoculated leaves and seedlings, respectively. The re-isolated fungus, was identical to D. eres based on morphological and molecular analysis, thus fulfilling Koch's postulates. To our knowledge, this is the first report of D. eres causing leaf blight of O. fragrans in China, supporting a basis for developing effective methods to manage this disease.

First report of Colletotrichum scovillei causing anthracnose fruit rot of pepper in Ohio.

In August 2019, the Ohio State University Vegetable Pathology laboratory received multiple bell and banana pepper fruits (Capsicum annuum, cvs. unknown) from Columbiana County, Ohio. The grower reported a disease incidence of 100% and severity of 70% in fruits across their pepper fields. Fruit lesions were brown, sunken, and covered with orange-colored sporulation. On banana peppers, the lesions mainly affected the blossom end of the fruits, while the lesions were distributed randomly on bell pepper fruits. Pieces of diseased tissue were cut from the fruit and surface sterilized in 0.5-0.6 % sodium hypochlorite, rinsed in sterile water, blotted dry, and placed on potato dextrose agar. All of the fungal cultures recovered were cottony, pale gray-green with shades of orange on the underside of the mycelial mat. Two representative isolates, SM209-19 (bell pepper) and SM210-19 (banana pepper), were grown on oatmeal agar to induce sporulation. Pink-orange concentric rings containing acervuli and conidia were present on the oatmeal agar plates after one week of growth at 22◦C (12-h dark/light). Conidia (n=29) were hyaline, aseptate, cylindrical in shape, and had an average length of 10.5 µm (std. dev. = 1.3 µm) and width of 4.1 µm (std. dev. = 0.6 µm) (Fig.1). DNA was extracted from both isolates using a DNeasy Plant Kit (Qiagen Inc, Germantown, MD), and partial sequences of the internal transcribed spacer (ITS) region, -tubulin 2 gene (TUB2), and glyceraldehyde-3-phosphate dehydrogenase gene (GDPH) were amplified by PCR with the following primers: ITS4/ITS5 (White et al. 1990), Bt1a/Bt1b (Glass et al. 1995), and GDF1/GDR1 (Guerber et al. 2003), and squenced. The ITS region of both isolates SM209-19 and SM210-19 (PP280815 and PP280816, respectively) showed 100% identity with C. scovillei (Cs) isolate LJTJ35 (KP748226). The partial sequences of GDPH, (PP320348, PP320349, respectively) showed 99% sequence identity with the Cs CBS 126528 (JQ948597) and 100% identity with Cs HP1 (MT826948) The partial sequences of TUB2 (PP472464 and PP472465, respectively) had 100% sequence similarity with the Cs HP1 and Cs CBS 126528 (MT826951, JQ949918 respectively). Pathogenicity was tested in a greenhouse experiment on blossoming bell pepper plants (cv. Carmen) by spraying 10 ml of 1 X 105 conidia/ml suspension onto flower blooms (nine plants per isolate). Control pepper plants were mock inoculated by spraying 10 mL of sterile deionized water. The plants were re-inoculated one week later. Brown, sunken lesions with orange sporulation developed on the fruits of inoculated plants 21 days after the initial inoculation (Fig. 2), while the mock-inoculated plants did not produce any symptoms. Culturing from symptomatic fruits on PDA, following the same method described above, produced fungal colonies with the same morphological traits previously described. C. scovillei causing anthracnose on pepper has been described in the US (Toporek et al. 2021), Brazil (Caires et al. 2014), China (Zhao et al. 2016), and different South Asia Countries (Khalimi et al. 2019). Open-field peppers are produced in Ohio on more than 5,400 acres, with a value of more than $53 million, with anthracnose being one of the most severe fungal diseases reducing yield. This newly reported Colletotichum species could represent a further threat for this crop. Further studies evaluating fungicide sensitivity and efficacy against this pathogen will be of crucial importance for disease management.

First report of Enterocytozoon bieneusi in Erinaceus europaeus and Hemiechinus auritus hedgehogs from recovery centers of Portugal.

Enterocytozoon bieneusi microsporidia are emerging pathogens infecting a wide range of vertebrate and invertebrate hosts, known to have zoonotic features since they infect both wild and domestic animals, and humans. Despite their significance, there is very limited epidemiological data on microsporidia in hedgehogs, especially European hedgehogs (Erinaceus europaeus) and long-eared hedgehogs (Hemiechinus auritus), the former known as synantropic hedgehogs, and the latter suited as pets. As such, the present study aimed to assess the presence of E. bieneusi in hedgehogs from Portugal. For this purpose, fecal samples from 110 hedgehogs of three species-E. europaeus (n=106), H. auritus (n=1), and Atelerix albiventris (n=3)-were collected and tested for E. bieneusi by PCR targeting the internal transcribed spacer region and the flanking small and large subunits of the rRNA. We found an overall occurrence of 22.7% (25/110; 95% confidence interval [CI]: 15.28-31.70), with 22.6% (24/106; 95% [CI]: 15.08-31.79) in E. europaeus, 100% (1/1) in H. auritus, and 0% in A. albiventris. Interestingly, three novel genotypes were identified, all belonging to the potentially zoonotic Group 1. Our findings highlight the importance of hedgehogs as potential reservoirs for E. bieneusi and emphasize the need for further research to understand their role in transmission dynamics and assess the associated risks to public and veterinary health.

Bionomic characterization of Anopheles mosquitoes in the Ethiopian highlands and lowlands

BackgroundThe protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands—resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands.MethodsHourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1).ResultsIn the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) (n = 1970, 41.9%), followed by An. demeilloni (n = 1133, 24.1%) and An. cinereus (n = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. (n = 1190, 36.9%), An. pretoriensis (n = 899, 27.9%), and An. demeilloni (n = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations.ConclusionsThe high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations.Graphical