Primers ITS4 Research Articles

First report of Curvularia leaf spot on Zea mays caused by Curvularia lunata in North Carolina.

In late July of 2022, corn leaves with spots were observed in a field at North Carolina State University's Vernon James Research and Extension Center (35.873294º N, 76.658599º W; Plymouth NC). The affected leaves contained small (two to three millimeters in diameter) tan-colored circular to oval lesions with reddish-brown margins and chlorotic haloes. The disease severity approached 50% on affected leaves. Leaf tissue from the margins of the lesions was excised and surface sterilized by soaking in 10 % bleach solution for two minutes followed by rinsing in distilled water. Next, leaf tissue was transferred to alkaline water agar amended with streptomycin (100 mgliter-1) and the plates were incubated at 27º C in dark. After one week, conidia were observed. Conidia were curved, light brown in color and contained three transverse septa. The size of the conidia (n= 30) ranged from 21-25 µm (average 24 µm) long by 7 to 10 µm wide (average 9 µm). These characteristics were typical of Curvularia lunata (Munkvold and White 2016). DNA from pure cultures was extracted, internal transcribed space region (ITS) was amplified using the primers ITS4 and ITS5 (White et al. 1990). Sequences were obtained and deposited into GenBank (accession no. OP998306). BLAST search indicated that the sequence was 100 % identical to C. lunata from Louisiana (MG 971305.1, Garcia- Aroca et al. 2018). Pathogenicity was confirmed on field corn cultivar 'Yellow dent' by inoculating two pots of two-week-old greenhouse-grown corn plants (6 plants per pot) by spraying conidial suspension with a concentration of 6 x 104 conidia /ml. The control plants (n= 6) were sprayed with distilled water and the plants were covered with a plastic bag for 24 hours. The plastic bags were removed and plants were moved to the greenhouse at 23º C. Four days after inoculation small round tan-colored lesions were observed and no symptoms on control plants. The experiment was repeated once and the pathogen was recovered from the symptomatic tissue. The morphology of the recovered isolate was similar to the original isolate. This is the first report of C. lunata causing leaf spot on corn in North Carolina. Although this disease has not been demonstrated to cause yield loss it has been detected more frequently in corn production areas of U.S. and should continue to be monitored going forward.

First report of Phytophthora gonapodyides causing root rot on raspberry in Canada.

Raspberry (Rubus idaeus L.) is an economically important fruit crop in Canada and about 80% of red raspberries are cultivated in British Columbia. In 2018, foliar symptoms associated with root rot and wilting complex disease were observed in raspberry field of Fraser Valley areas of British Columbia. Plants were stunted with reduced numbers of primocanes. Chlorosis and necrosis on leaves and partial wilting of branches were observed. When plants were uprooted, necrosis and browning on roots were observed. Two isolates of oomycetes pathogen were isolated using baiting with rhododendron leaves and pear fruit as described in Sapkota et al. 2022. Using FastDNA Spin kit (MP Biomedical, Burlingame, CA), genomic DNA of pathogen isolates was extracted from mycelia cultured on 20% clarified V8 agar medium amended with 10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin (V8PAR) per liter following the manufacturer's standard protocol. Pathogens were identified using colony morphology on 20% clarified V8 PAR as well as internal transcribed spacer (ITS) sequencing with ITS1 primers (White et al. 1990) and multiplex targeted-sequencing with degenerate primers of three nuclear genes: heat shock protein90 (HSP90), elongation factor 1 alpha (EF1α) and beta tubulin (βtub). BLAST searches of ITS sequences of isolates of this study (accession nos. OP180065, OP180066) in NCBI GenBank showed 98.5 to 99.6% identity with the ITS sequence of P. gonapodyides (accession nos. MN513238.1, MG753496.1). Multiplex targeted sequencing also identified both isolates as a P. gonapodyides (accession nos. SRR20227809, SRR20227807) when mapped with the reference sequences (accession nos. HSP90: KX251233.1, EF1α: KX251231.1, β-tub: KX639710.1). Pathogenicity was confirmed by inoculating mycelial suspension of one isolate of P. gonapodyides on root of intact plants and mycelial plugs of two isolates on detached stems of the raspberry plants, 'Chemainus' in the greenhouse using methods described in Sapkota et al. 2022. Two experiments were conducted with three replicates in each test. Experiments were arranged using completely randomized design. In detached stem assays, distinct dark-lesion symptom appeared at 7 to 9 days after inoculation while uninoculated control stems remained asymptomatic. Intact plants showed wilting and foliar symptoms 15 days after inoculation and progressed higher at 4 to 5 weeks after inoculation. Root infection with dark brown to black color was observed when roots were assessed at 5 weeks after inoculation. The diseased root and crown tissues tested positive for Phytophthora in Agdia ImmunoStrip and P. gonapodyides was re-isolated and confirmed with multiplex-targeted sequencing. Phytophthora gonapodyides was previously reported from raspberry in Chile (Wilcox and Latorre 2002). To our best knowledge, this is the first report of P. gonapodyides infecting red raspberry in British Columbia, Canada. The detection of new Phytophthora species on raspberry may become a new potential problem to growers in addition to P. rubi, which is already a major cause of raspberry decline in the region.

Improvement of Methods for Safety Control of Microbial Producers and Food Produced Therewith.

The study substantiates the necessity to implement the algorithm of molecular-genetic assessment of biosafety of the genetically modified microorganisms (GMM) and to develop standardized methods to test the genetically modified strains producing enzymes, bioactive substances, and other products of microbial synthesis prior to their use in food industry. Analysis of microbial producers and related food products for the presence of GMM-associated DNA revealed high incidence of the marker genes amp and lacZ in enzyme preparations and in mycelium of industrial genetically modified producer of Aspergillus genus. The procedure of extraction of DNA from mycelium of mold fungi is optimized by including the stage of additional purification of the extracts, assessment of their purity by PCR with universal ITS primers, and determination of effective DNA concentration in the samples prior to conduction of the molecular genetic assay. For identification and genotyping of mold fungi (the biotechnological producers of enzyme preparations), the Sanger sequencing method was adapted. Using this modified method, we determined the species of five equivocally identified strains of Aspergillus genus. To identify the closely-related micromycetes of Ascomycota division, a genotyping algorithm was developed based on amplification of total DNA with expanded panel of primers and DNA sequencing by capillary electrophoresis.

First Report of Rust Disease on Alcea rosea Caused by Puccinia modiolae in Korea.

Alcea rosea, in the family Malvaceae, is a biennial plant native to China and is grown typically for gardening in Korea (Lee 2003). Seven microcyclic Puccinia species have been reported on A. rosea: P. heterogenea, P. heterospora, P. lobata, P. malvacearum, P. platyspora, P. sherardiana, and P. modiolae (Demers et al. 2015; Aime and Abbasi 2018). In early May 2022, characteristic symptoms of rust were observed on four of ten seedlings of A. rosea purchased at a wholesale nursery (36°50'19.8″N, 128°55'28.7″E) in Bonghwa, Korea. Rust spots were present on almost 90% of the 1,000 seedlings of A. rosea in that nursery during our survey in late May. Through a distribution survey from June to July 2022, similar symptomatic leaves were additionally collected from the leaves of A. rosea grown in gardens at five sites in Gimcheon (two sites), Gumi (one), Seongju (one), and Busan (one). Spots were yellow-orange the center surrounded by chlorotic haloes on the adaxial leaf surface, and reddish-brown or dark brown pustules on the abaxial leaf surface. Over time, the spots enlarged and coalesced, causing the decay of large sections of the leaves, and heavily infected leaves fell early. Spermogonia, produced at the center of the chlorotic spot on the adaxial leaf surface, were subepidermal, obovoid, and 113.2-164.5 × 97.6-153.3 μm in size. Telia were reddish-brown to dark brown, round, mostly grouped, 0.28-0.61 mm in diameter, and mainly formed on the abaxial leaf surface but sometimes on the adaxial leaf surface also. Teliospores were two-celled, but rarely one- or three-celled, and were fusoid and 37. 6-110 × 12.4-21.5 μm in size; the wall was yellowish or almost colorless, smooth, 1.2-2.6 μm thick at the sides, and up to 7.4 μm thick at the apex. The morphological characteristics were similar to those of P. modiolae, although the teliospores in our study were longer than those observed by Aime and Abbasi (2018). For phylogenetic analysis, genomic DNA was extracted from the teliospores of each regional specimen. Partial 18S, internal transcribed spacer (ITS), and partial 28S sequences were amplified using primers NS1, ITS4, ITS5, and LR11. The PCR products were sequenced (Celemics, Seoul, Korea) and deposited in GenBank. The ITS-partial large subunit (LSU) sequence and 28S sequences had 100% homology with other P. modiolae sequences deposited in GenBank (accession numbers are shown in Fig. 2). In the phylogenetic trees of the ITS and LSU sequences, the isolates collected in this study were grouped with the reference sequences of P. modiolae, including the Korean isolate (ON631218) recently reported on Malva verticillata by Lee et al. (2022). For the pathogenicity test, the teliospores with germinating basidiospores were suspended in sterile distilled water and smeared on the upper surface of asymptomatic A. rosea leaves in August. The inoculated plants were sprayed with distilled water and kept in the dark with saturated moisture for 24 h in an isolated glass house of the Animal & Plant Quarantine Agent. After 2 weeks, typical rust spots and telia of P. modiolae were observed on the leaves of the inoculated plants, but not in the control plants, which were only sprayed with distilled, sterilized water and otherwise treated similarly to the inoculated plants. The results of this study show that the casual fungus is P. modiolae, which has been commonly found in A. rosea in Korea. To the best of our knowledge, this is the first report of P. modiolae in A. rosea in Korea.

Shift in fungal communities associated with Pinus sylvestris stands affected by root rot

Introduction
 Scots pine (Pinus sylvestris) covers large areas in European regions with significant economic importance to Ukrainian forest industry. Root rot caused by the wood-decay fungus Heterobasidion annosum damages both below- and above-ground parts of Scots pines. The disease progress is likely to be affected by reshaping in the forest such as soil properties, vegetation composition, and tree age. These changes are apparently followed up by paralleled shifts in fungal community composition on forest soil with potential feedback on ecosystem functioning.
 The objective of the study was to screen fungal groups associated with the root system of P. sylvestris in stands affected by H.annosum s.s. to better understand the pathogenesis and development of root rot infection, as well as to recognize whether root size and disease severity affect diversity of fungi of the root system in the forest-steppe conditions of Ukraine. The additional object was to study other resident microflora of P. sylvestris root infested by H.annosum s.s. to find out whether the H. annosum s.s. impacts the overall diversity of other fungi.
 Materials and Methods
 The field study was carried out in 2018–2020. Field study sites were pure pine forest stands located in Kharkiv region (compartment 126, subcompartment 7, tract Bugri, Kharkiv Forest Research Station). Wood core and root samples from P. sylvestris were collected from the five infected (50–100 m apart from each other) and five non-infected trees (up to 500 m apart from the infested area and 50–100 m apart from each other). Wood and root samples were used for fungal culturing and direct sequencing using ITS1F and ITS4 primers.
 Results
 In the present study, we tried to evaluate fungal communities across diseased Pinus sylvestris stands and investigated correlations between taxonomic composition and forest health. Not surprisingly, root rot infestation had a significant effect on root-associated fungal abundance and diversity. During disease development, the root-associated fungal community shifted in composition from dominance by saprotrophic fungi to ectomycorrhizal and pathogenic fungal species. Our results suggested that maintenance of functional diversity in the root-associated fungal community may sustain long-term forest health or even root rot resistance to some extent by retaining a capacity for symbiosis-driven recycling of organic nutrients; however, this hypothesis is necessary to carefully examine and prove further.
 Conclusions
 Fungal culturing from 10 surface-sterilized wood cores resulted in 21 fungal cultures, 2.1 per wood segment. Direct sequencing from 40 surface-sterilized segments of lateral roots resulted in 247 fungal sequences or 6.2 per root segment on average. The most dominant fungi from the infested trees of Pinus sylvestris were Dactylonectria macrodidyma (4.98%), Acremonium sp (4.52%), Cladosporium cladosporioides (4.07%) from Ascomycota and Heterobasidion annosum s.s. (4.07%) from Basidiomycota, while for non-infested group Unidentified Ascomycota175244 (13.19%), Penicillium spinulosum (9.89%), Acremonium sp. (8.79%), Bionectriaceae sp. (8.79%) were the most common.
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Morphology and molecular identification of Candida Species isolated from oral pediatric

In newborns, Candida is responsible for the common oral thrush and rash in skin folds and in the diaper area. Before the advent of modern sanitary measures and topical antifungal treatments, infants died from dehydration due to severe oral mucocandidiasis. Oral thrush is more likely to occur in infants and older adults due to reduced immunity. The study aimed to shed light on the relationship between Thrush and Candidia and To molecular identification of Candidia spp. isolated from Thrush. There are 100 samples of Oral swabs collected from pediatric with Thrush at ages ranging from (1day-12 years). All samples cultured on SDA, MSA and MEA. Several tests were used to identify the types of isolated yeasts, including growth on Chromogenic agar medium, as well as biochemical and molecular tests using primers ITS1, ITS4. The Results found that a six species of Candida were isolated on Chromogenic: C. albicans, C. glabrata, C. tropicalis, C. dubliniensis, C. krusei and C. kefyr, Also, showed the number of yeasts that were genetically diagnosed by molecular diagnosis was five species of yeasts as follows 2 isolates C. tropicalis from 22% and one isolate for each of the following types C. albicans, C. kefyr, C. dubliniensis and C. spp with 11% of all isolates where C. albicans showed the highest percentage. The results of the statistical analysis of the isolates showed that the most affected age group of pediatric was (month-1 year) in the rate of 24%. Antifungal susceptibility was studied, as the results indicated that all Candida isolates were sensitive to itraconazole, while all Candida isolates were resistant to fluconazole Furthermore, most types of Candida were resistant to Nystatin and Amphotericin B, except for three type’s C. krusei, C. glabrata, and C. kefyr. The most common pathogen in pediatric is Candida spp. In pediatric, the results indicated that the best treatment to eliminate Candida Itraconazol, Clotrimazol, Nystatin, and Amphotricine B Nystatin respectively

First Report of Brown Rot Caused by Monilinia polystroma on Sweet Cherry and Almond in Italy.

Brown rot decay is an important disease of pome and stone fruits. In Italy, the main pathogens on stone fruits are Monilinia laxa, M. fructigena, and M. fructicola (Spitaler et al. 2022a). In addition, Monilinia polystroma (G. Leeuwen) L. M. Kohn (van Leeuwen et al. 2002), was recently found in Italy on peach (Martini et al. 2014), pear (Martini et al. 2015), plum (Abate et al. 2018), apple (Rosati et al. 2021), and quince (Spitaler et al. 2022b). In South Tyrol province, sweet cherry (Prunus avium L.) and almond (Prunus dulcis Mill. D. A. Webb), plants of the Rosaceae family and belonging to stone fruits, were observed to be frequently affected by brown rot. Affected cherries as well as almonds showed brown lesions, covered by yellowish or buff-colored stroma in concentric rings. Symptomatic cherries became shriveled, while symptomatic almonds remained firm. To determine the pathogen, single spore isolates were obtained from five symptomatic fruits, each from a cherry orchard of the cultivar Kordia in July 2021 and almond trees of the variety Dulcis in August 2021. Both sample sites were situated in Vadena/Pfatten. Infestation in the cherry orchard, covered by a rain-protection foil, was determined to be about 1 %. In almond, over 50 % of the fruits of various ripening stages showed brown rot symptoms. On potato dextrose agar (PDA) at 22 °C and a photoperiod of 16 h, isolates from both fruits matched the morphological characteristics of M. polystroma (Vasić et al. 2016) within 14 days. DNA was extracted from mycelium and the rRNA encoding gene region using ITS4 and ITS6 primers as well as a genomic sequence of unknown function using the primers UniMon_Forw and UniMon_Rev (Petróczy et al. 2012), were amplified and sequenced. MegaBLAST analysis revealed 100 % identity with M. polystroma sequences of the NCBI GenBank (rRNA encoding region: NR_154198; genomic region: JN128836). Sequences were deposited in GenBank under the accession numbers OP642545/OP654171 (cherry) and OP642546/OP654172 (almond). Pathogenicity was confirmed with mature cherries cultivar Duroncino or almost mature almond fruit of the variety Dulcis, respectively: 16 samples each for both fruits were surface-sterilized by dipping in 75 % ethanol for 10 s and subsequent rinsing with sterile water for 10 s. Mycelial plugs (1 mm) were dislodged from a 7-day old colony and inserted in a 1 mm hole into the fruits. Incubation was performed in plastic boxes under the conditions described above. PDA-inoculated fruit were used as controls. All cherries and all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation, respectively. Control fruits remained symptomless. Conidia were produced in branched chains on mycelium-inoculated fruit. Conidia were one-celled, limoniform, hyaline, measuring 13.1 to 22.2 × 9.7 to 14.8 µm (cherry) and 14.1 to 20.8 × 10.7 to 15.3 µm (almond). Additionally, 16 fruits each were inoculated with 20 µL conidial suspension (5 x 10^3 spores/mL) from mycelium-inoculated fruits. All cherries as well as all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation. Control fruits remained symptomless. To confirm identity, the fungus was isolated from five spore-inoculated fruits each for cherry and almond. The isolates showed identical morphological features and sequence identity as the original isolates. To our knowledge, this is also the first report of M. polystroma on almond, while the pathogen has already been reported on sweet cherry in Poland (Poniatowska et al. 2016). These additional host plants identified in this study suggest a broad impact of M. polystroma on Italian stone fruit production. Due to the economically important cultivation of stone fruit, further knowledge about the pathogens' host range will help to assign brown rot symptoms to M. polystroma and to improve targeted control strategies.

Isolation and Molecular Characterization of Collar Rot Causing Pathogen Aspergillus niger on Groundnut in Rajasthan, India

Groundnut, a major pulse crop also grown as an oil seed over 90 countries worldwide, is important to the Indian economy. The states of Andhra Pradesh, Gujarat, Rajasthan and Tamil Nadu are the main producers of groundnut. In 2021, disease incidence with symptoms similar to collar rot was reported from Jaipur district of Rajasthan. The fungal strain was isolated from the collar region of susceptible groundnut variety TAG-24. White colonies with mycelia were observed in 3-4 days which eventually turned dark brown in color. Pathogenicity tests were performed on healthy groundnut and collar rot symptoms developed within 7-8 days of inoculation. Sequencing of PCR products amplified with the18S rRNA (NS1F-NS8R) and ITS (NS1F-ITS4R) primers indicated 99.4% and 100% identity respectively with a reference strain of Aspergillus niger CBS 554.65 (ATCC 16888) in the NCBI-Genebank database. Based on the morphological and molecular characteristics, the fungal pathogen was identified as Aspergillus niger and submitted to Gene Bank with the accession nos. ON954789 and OQ653131. The the culture is submitted to NCMR-NCCS (accession number MCC 9892).

Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India

Background: Fusarium wilt in lentil (Lens culinaris L.) infected by Fusarium oxysporum is considered as one of the major biotic factors for low productivity in lentil. The present study was conducted to determine nine Fusarium isolates (W1-W8 and W10) which were isolated from different lentil growing fields of West Bengal, India and were characterized for their physio-morphological, biochemical and molecular traits in vitro. Methods: Nine Fusarium infected lentil plant samples were collected from 9 different places of West Bengal. Pathogen was isolated from lentil samples using PDA media followed by cultural, morphological, pathogenic and molecular variability studies for all the isolates of Fusarium. Type of pigmentation, sporulation, conidia size of each isolate was recorded by observing culture plate after complete growth of the mycelium. Pathogenicity test were done to check the aggressiveness of all the 9 isolates of Fusarium in three varieties of lentil (Maitri, HUL-57 and BM-6). The isolated fungal DNA was amplified with specific primer pair, Forward primer ITS 1 and Reverse primer ITS 4. Result: Morphological assessment of 9 isolates showed distinct identification characters and variability. The mycelia of all the isolates were septate, profusely branched with differential pigmentation, texture and margin.All the Fusarium isolates produced micro-and macro-conidia in pure culture within seven days after inoculation and showed different morphological characters in pathogenicity test, variety Maitri showed better germination (100% in W2, W4, W6, W7, W8 and W10) and less disease severity against most of the strains (75% in W1) as compared to other two varieties against and hence variety Maitri taken into consideration for greenhouse and field trials. Furthermore, identification of molecular variability was performed by sequence analysis of rDNA-ITS region of each isolate and sequencing of rDNA-ITS region supported the morphological study and confirmed the associated fungi at species level.

Identification of Resistant Genotypes and Integrated Management of Dry Root Rot of Chickpea

Background: Dry root rot is an economically important soil borne disease of chickpea in India. The pathogen, Rhizoctonia bataticola, is a soil borne fungus resulting in significant losses in yield. Therefore, the present investigation was aimed to identify the management strategy of disease through identification new sources of resistance and integrated management of disease. Methods: R. bataticola was isolated and purified by using hyphal tip technique and molecular detection was done by using ITS primers. One hundred chickpea germplasm entries were screened under in both field and advanced phenotyping in glass house. The field trials on integrated management of the disease were conducted with different treatments using randomized block design. Result: Fourteen resistant and five moderately resistant genotypes were identified under artificial epiphytotic conditions. Advanced screening of these 19 genotypes under phenotyping technique yielded four resistant and five moderately resistant genotypes. Among nine treatments, seed treatment with mancozeb 50% + carbendazim 25% WP @ 3.5 g/kg followed by soil drenching of mancozeb 50% + carbendazim 25% WP @ 3 g/l water to infected and surrounding plants was found highly effective by recording least disease incidence with highest seed yield, test weight and benefit cost ratio.

Molecular Identification and Genetic Diversity of Alternaria Isolates Causing Leaf Spot Disease in Cotton from Major Cotton Growing Areas of South Zone of India

Background: Alternaria leaf spot caused by two species namely Alternaria macrospora and Alternaria alternata is an important foliar disease of cotton. Conidial morphology showed that most of the isolates in the study belonged to A. macrospora. Molecular confirmation is necessary to strengthen the identification of species in Alternaria. Genetic diversity study of Alternaria isolates using ISSR and hyper variable SSR primers will provide variation and grouping among the isolates collected from major areas of South Zone of India. Present study was conducted to identify Alternaria isolates at species level using molecular methods (species specific primers) and genetic diversity analysis using ITS, SSR and ISSR primers. Methods: Reported species-specific primers such as AmF and AmR as well as AaF2 and AaF3 were used for Alternaria species identification. ITS region was amplified through ITS1 and ITS4 and sequences were used for identification and clustering of isolates. Thirteen hyper variable SSR primers specific to Alternaria were designed based on the sequences retrieved from NCBI and used for diversity study. Six different ISSR primers were also used for genetic diversity study. Result: Reported species-specific primers found not suitable to identify A. macrospora and A. alternata at species level. Two SSR primers were found to be effective in showing variability among the isolates. Six clusters were formed at 71 percent genetic dissimilarity among 15 isolates of Alternaria through ISSR primers. Five clusters were formed in ITS sequence’s diversity analysis. Blasting of ITS sequences of 15 selected isolates at NCBI showed that all belong to A. alternata. This was due to absence or presence of very few sequences of A. macrospora in NCBI database itself. Further house-keeping genes like Alt a1, Plasma membrane ATPase, GAPDH and TEF -1 α sequence analysis will be useful for confirmation of A. macrospora at species level.

Molecular Identification and Biotechnological Potential of Cerithidea cingulata-and Lottia scabra Associated Fungi as Extracellular Enzyme Producer and Anti-Vibriosis Agent

Mangroves are complex, unique, and dynamic ecosystems that host organisms that have special physiological adaptations to fluctuations in salinity, temperature, and pH. Gastropods have an important role in the mangrove ecosystem for food cycles and webs by helping to provide nutrients and micro-habitats for microbes. Micro-fungi isolated from mangrove ecosystems are productive sources of enzymes and bioactive compounds such as antibacterial and antifungal. In this study a explored the enzymatic and anti-vibrio activity of fungi associated with the marine gastropod Cerithidea cingulata and Lottia scabra. collected in three mangrove forests in North Sulawesi, Indonesia. The fungi associated with the specimens were inoculated on malt extract agar (MEA) using the tapping method. Enzymatic and anti-vibrio assays were conducted applying the plug method. Positive effects of the fungal compounds on Vibrio were indicated by clear zones formed around the isolates. A total of 13 fungal species were isolated from C. cingulata (19MB-C5 and 19MBa-A4) and L. scabra (19MT-07 and 19MT-18). Most fungal isolates have moderate growth rate. The isolate with highest cellulolytic, proteolytic, and anti-vibriosis activity (Vibrio parahaemolyticus, V. harveyi, V. vulnificus, and V. alginolyticus), derived from C. cingulata 19MB-C5 was closely related to Aspergillus niger, as revealed by molecular analysis using the universal primers ITS1 and ITS4 (similarity value 99%). A. niger 19MB-C5-3 has potential as a bioremediation and anti-vibrio agent that can be applied in aquaculture.

Molecular Detection of Fusarium species infected Corn in Kurdistan region-Iraq

Corn or Maize product is regarded as one of the essential products in the world and stands third product after the rice and wheat crops. Different fungal pathogens attack corn plants; one of them is ear rot, brought on by Fusarium species and whose occurrence is primarily influenced by environmental factors. In order to isolate and identify Fusarium species from corn plants and their prevalence, 50 samples of corn were collected during September, October, and November of 2021 from 30 corn fields in 14 regions of different places in the Kurdistan region of Iraq. From all samples, 39 isolates of Fusarium were detected and based on morphological characteristics, six other species of Fusarium were identified, namely F. verticillioides (33.34%), F. proliferatum (25.64%), F. oxysporum (12.82%), F. incarnatum and F. equiseti (10.25% each), then F. fujikuroi (7.7%). The most prevalent species was F. verticillioides which was isolated from seven corn fields and significantly higher than all other isolated species. All Fusarium isolates were also molecularly identified depending on amplifying the internal transcribed spacer (ITS) universal region using forward ITS1 and reverse ITS4 primers and indicated DNA fragments ranged from 550 to 570 bp. The PCR fragments of the amplified ITS region were sequenced, aligned and registered in NCBI GeneBank with specified accession numbers. The phylogenetic tree and all analyzes were performed using the MEGA program version 11.0.13. The current study concluded that the corn fields in the Kurdistan region are infected with different Fusarium species, and the most common species is F. verticillioides. As well as the Fusarium species in the Kurdistan region have close evolutionary history to the same species in other countries. Thus, the study recommends more research to investigate the occurrence of toxigenic Fusarium species associated with cereal grains in the region.

Management of Fusarium Wilt Disease of Maize (Zea mays L.) with Selected Antimycotic Plant Extracts and Inhibitory Bacillus Strains.

Maize (Zea mays L. var. Everta) is an economically significant crop cultivated worldwide. Unfortunately, microbial diseases, especially, mycopathogens such as Fusarium species, militate against the production of maize. Protective microbial species and bioactive plant extracts have been studied in the control of plant pathogens. However, there is a dearth of information on their comparative efficacy and effect on wilt disease of maize caused by Fusarium solani, as investigated in this study. Using the ITS and 16s rDNA primers for fungal pathogen and bacterial strains, respectively, the mycopathogen was confirmed as Fusarium solani FCI20, while biocontrol Bacillus strains were identified as Bacillus velezensis EBs02 and Bacillus thuringiensis EBs04. Fusarium solani FCI20 successfully infected maize seedlings through rhizosphere inoculation, and caused severe leaf chlorosis, necrosis and wilt in maize seedlings. Bacillus thuringiensis EBs04 expressed the highest in vitro mycelial inhibition (85.20%), followed by Gmelina arborea (78.58%), while Milicia excelsa appeared to express the lowest mycelial inhibition potential (49.95%). Bacillus velezensis EBs02 expressed the highest in vivo disease severity reduction in maize seedlings (with percentage disease control of 84.16), while B. thuringiensis-treated plants had the lowest incidence of wilt disease (4.32%). However, contrary to their in vitro mycelial inhibition potential, B. velezensis EBs02, Gmelina arborea, Milicia excelsa and Cola nitida expressed radically different levels of in vivo wilt disease control in maize seedlings. Consequent upon the biocontrol patterns observed in this study, in vivo assay should be considered in the preliminary selection of promising biocontrol agents against phytopathogens, such as Fusarium species.

First report of Diaporthe tulliensis causing leaf spot of betel vine

The betel vine (Piper betle) is grown in protected areas under the humid and shaded conditions required for the plant's growth. This damp, shady environment favours the spread of numerous diseases, particularly leaf spot disease (Chattopadhayay & Maity, 1990). During 2019–2022, diseased betel leaves were collected from the East Khasi Hills and Ri Bhoi districts of Meghalaya, India. The leaves had spots that were irregular in shape and size, light to dark brown and surrounded by a chlorotic yellow halo, which gradually spread to the leaf centre followed by yellowing, drying and drooping of the whole leaf. An area of approximately of 8 m2 was affected and 5–20% of the plants had symptoms, depending on the year. The number of leaf spots increased with temperature and humidity. To identify the causal agent, lesions (5-10 mm in diameter) were cut from ten infected leaves (Figure 1), surface disinfected with 70% ethanol, rinsed three times in sterile distilled water and then plated on potato dextrose agar (PDA) amended with 30mg/ml of streptomycin sulphate. The same colony type was obtained from all leaves which was white producing yellowish-green pigments and the reverse olive green to brown after seven days (Figure 2A-B) and after 30 days of incubation at 30°C, produced semi-immersed black pycnidial conidiomata which exuded pale yellow to black conidial droplets (Figure 2C). The α-conidia were one to two-celled, hyaline, oval to fusiform, guttulate and 5.3 × 2.5 μm (Figure 2D), conidiophores hyaline but no β- and γ-conidia were observed (Li et al., 2022), and conidiogenous cells were hyaline and septate (Figure 2E-F). The identification of the fungus was confirmed by sequence analysis using ITS1 and ITS4 primers (White et al., 1990) (GenBank Accession No. OQ402579). BLAST analysis showed 100% identity with sequences of Diaporthe tulliensis (e.g. MN911384). In phylogenetic analysis, the isolate in the current study clustered with D. tulliensis using the neighbour-joining method, showing 100% identity (Figure 3). To confirm pathogenicity, the isolate was grown in potato dextrose broth at 28°C for seven days, then inoculated onto five healthy betel leaves using pin-prick inoculation with 10 μl spore suspension (1×105 spores/ml). The untreated control did not show any symptoms, however, the inoculated plants had brown necrotic spots followed by yellowing after four days (Figure 4). On PDA, cultures isolated from infected plant leaf lesions exhibited the same morphology as D. tulliensis and similar conidia were discovered during microscopic analysis subsequently satisfying Koch's postulates. To the best of our knowledge, this is the first report of D. tulliensis as a fungal pathogen of betel leaves. Diaporthe tulliensis has been reported to cause leaf spots on coffee in China (Gorg et al., 2020) and on Parthenocissus tricuspidata in Taiwan (Huang et al., 2021), cacao pod rot in Puerto Rico (Serrato-Diaz et al., 2022), and stem canker of kiwifruit in China (Bai et al., 2017) and of jasmine in Taiwan (Hsu et al., 2023). The authors thank the Head of the Department of Botany, North-Eastern Hill University for providing laboratory facilities. The first author is also grateful to the Non-Net Fellowship, New Delhi, for financial support in the form of a research fellowship.

PD19-08 THE URINARY FUNGAL MICROBIOME IN THOSE WITH OVERACTIVE BLADDER IS LESS ABUNDANT IN MALASSEZIA AND MORE ABUNDANT IN ASPERGILLUS RELATIVE TO HEALTHY CONTROLS

You have accessJournal of UrologyCME1 Apr 2023PD19-08 THE URINARY FUNGAL MICROBIOME IN THOSE WITH OVERACTIVE BLADDER IS LESS ABUNDANT IN MALASSEZIA AND MORE ABUNDANT IN ASPERGILLUS RELATIVE TO HEALTHY CONTROLS Glenn Werneburg, Daniel Hettel, Madison Lyon, Ava Adler, Peace Orji, Suruchi Ramanujan, Jacqueline Zillioux, Bradley Gill, Howard Goldman, Sandip Vasavada, and Aaron Miller Glenn WerneburgGlenn Werneburg More articles by this author , Daniel HettelDaniel Hettel More articles by this author , Madison LyonMadison Lyon More articles by this author , Ava AdlerAva Adler More articles by this author , Peace OrjiPeace Orji More articles by this author , Suruchi RamanujanSuruchi Ramanujan More articles by this author , Jacqueline ZilliouxJacqueline Zillioux More articles by this author , Bradley GillBradley Gill More articles by this author , Howard GoldmanHoward Goldman More articles by this author , Sandip VasavadaSandip Vasavada More articles by this author , and Aaron MillerAaron Miller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003285.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The urinary tract harbors its own microbiota, but fungi composition remains poorly characterized. Overactive bladder (OAB) is a common urologic diagnosis, with predominant symptoms being urinary urgency and frequency. We sought to investigate the urinary fungal microbiome of the overactive bladder population and compare that with healthy controls. We hypothesized that the population with OAB would harbor unique patterns of fungi relative to healthy controls. Understanding the fungi associated with OAB could open new avenues toward the investigation of fungi in disease manifestation and treatment response. METHODS: Female patients age 18 and over with overactive bladder by AUA/SUFU criteria were included, as were a group of control adult female patients without urologic diagnoses. Participants provided a clean catch urine specimen. Samples were subjected to next-generation sequencing using ITS primers to capture fungii, and fungal diversity was determined. Diversity and taxonomic profiles were compared between groups by two-tailed t-tests and PERMANOVA. RESULTS: 46 patients with OAB and 32 healthy controls were included. The OAB group median age was 65.7 (IQR 14.3), and the healthy control group median age was 60.4 (IQR 38.6) and. Fungi were detected in all urine samples. The predominant phylum was Ascomycota in overactive bladder patients and Basidiomycota in healthy controls. Malassezia taxa was less (12% OAB, 18% other, p<0.05) abundant in the overactive bladder group and Aspergillus was more abundant in the OAB group (14% OAB versus 7% healthy, p<0.05). CONCLUSIONS: The urinary fungal microbiome, or “mycobiome” differed significantly between the selected population with OAB and a healthy control population. Ascomycota was the predominant phylum. Malassezia was less abundant in those with OAB and Aspergillus more abundant. Additional study is needed to further delineate the relationship between the fungal microbiome and OAB, whether it may be a cause or effect of the OAB symptom complex, and whether it can be modulated to improve OAB treatment. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e580 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Glenn Werneburg More articles by this author Daniel Hettel More articles by this author Madison Lyon More articles by this author Ava Adler More articles by this author Peace Orji More articles by this author Suruchi Ramanujan More articles by this author Jacqueline Zillioux More articles by this author Bradley Gill More articles by this author Howard Goldman More articles by this author Sandip Vasavada More articles by this author Aaron Miller More articles by this author Expand All Advertisement PDF downloadLoading ...

First report of Choanephora rot on Lettuce (Lactuca sativa L.) caused by Choanephora cucurbitarum in Korea.

Lettuce (Lactuca sativa L.) is a vegetable belonging to the family Asteraceae, and one of the major leafy vegetables in Korea. Lettuce has long been consumed as a fresh vegetable. In 2020, 96,774 tons were produced in about 3,800 ha according to Statistics Korea, and various types of lettuce have been bred for year-round production in line with consumer needs (Jang 2007). In August 2020, symptoms of a disease on young leaves of green lettuce (cv. Cheongpungyeoreumchima) were observed in the commercial greenhouse, Jeongeup (35°37'17.5"N, 126°53'40.4"E), Korea. About 60% of plants in 500 m2 of the cultivation area were infected. The tips of infected leaves appeared water-soaked, with brown to black lesions and were covered with dark masses of sporangiophores. Two fungal isolates NC20860 and NC20861 were isolated from monosporous sporangiola in infected leaves. Two isolates produced fast-growing colonies on potato dextrose agar medium (PDA) with abundant white mycelium at the top and bright yellow pigmentation at the underside in only 2 days. Morphological characteristics of the two fungal isolates were investigated from 30 days after inoculation on PDA. Sporangiophores bearing sporangia were erect, non-septate, unbranched, hyaline, and straight. Sporangia were hyaline to brown, globose to subglobose, 28.4 to 33.6 µm diameter. Sporangiospores from sporangia were brown to dark brown, ellipsoid to ovoid, distinctly longitudinally striate, with hyaline appendages at each pole, 6.3 to 7.5 µm wide and 13.0 to 16.0 µm long. Sporangiophores bearing sporangiola were erect, unbranched, hyaline and sporangiola were fusiform, ellipsoid to broadly ellipsoid, 7.6 to 9.8 µm wide, 13.3 to 17.8 µm long. Based on the morphological and cultural characteristics, this fungus was identified as Choanephora cucurbitarum (Berk.&Ravenel) Thaxt (Kirk 1984). To identify the species of the two isolates, DNA was extracted by using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). The internal transcribed spacer (ITS) region of ribosomal DNA was amplified using nTaq-Tenuto polymerase chain reaction (PCR) kit and therein specified protocol (Enzynomics). The primers ITS1F and ITS4 were used for PCR and sequencing (Gardes 1991). The ITS sequences of the isolates NC20860 and NC20861 from Korea (NCBI GenBank accession no.MZ960299, MZ960300) and the sequences of C. cucurbitarum strains CBS 150.51 (MH856791.1), and KUS-F29113 (KU316934.1) were compared. The sequences of the isolates were identical to those of the reference strains of C. cucurbitarum CBS 150.51 (reference/isolates base pairs, 566/566) and KUS-F29113 (572/572) at the 100% of sequence similarity. Pathogenicity test was conducted using seedlings of 8-week-old green lettuce (cv. Cheongsimchima) by spraying spore suspension (2⨉104 spores/ml). The plants were covered with plastic bags and incubated at 25 ℃ and 70 to 90% RH with a 12-h photoperiod for 20 days. The symptoms were observed only on the inoculated plants within 20 days after inoculation. The inner leaves of the inoculated lettuces had water-soaked lesions. No symptoms were observed in controls. C. cucurbitarum was reisolated from inoculated lettuce, fulfilling Koch's postulates. This fungus has been reported as the causal pathogen of fruit and blossom rot in cucurbits and other plants (Akwaji 2014). However, to our knowledge, occurrence of this pathogen on lettuce has not been reported as yet. This disease is crucial for producers because infected leaves directly results in yield loss of lettuce. This is the first report of Choanephora rot on lettuce caused by C. cucurbitarum in Korea and worldwide.

First report of Phytophthora hibernalis, P. multivora and P. niederhauserii causing root rot and bleeding cankers on Eucalyptus globulus in Portugal

During spring 2022 Phytophthora related-disease symptoms were observed in three Eucalyptus globulus plantations in central Portugal (Aveiro district). Affected trees displayed a range of symptoms including root rot, bleeding cankers and extensive canopy dieback (Figure 1). Average disease incidence and tree mortality rate were estimated as 67 and 20% along three linear transects of 50 m, respectively. In order to isolate the causal agents, rhizosphere and root samples were collected from 20 diseased trees chosen at random in the monitored plantations. Samples were processed as described by Bregant et al. (2023) and cultured on carrot agar at 20°C in the dark. Phytophthora colonies were examined and identifying features recorded and compared with published morphological records of Phytophthora species. The following three species were identified: Phytophthora multivora (12/20 isolates), P. hibernalis (6/20) and P. niederhauserii (2/20) (Figure 2). The identity of all isolates was confirmed by analysis of the internal transcribed spacer region using the universal primers ITS1 and ITS4. DNA extraction, PCR amplification and sequencing were performed according to Bregant et al. (2023). A BLASTn search against the GenBank database showed 100% identity with sequences of the ex-type cultures of P. hibernalis (CBS 114104), P. multivora (CBS 124094) and P. niederhauserii (WPC P10616). The ITS sequence of a representative isolate of each species was deposited in GenBank (P. hibernalis: GenBank Accession No. OQ520314; P. multivora: OQ520315; and P. niederhauserii: OQ520316). The pathogenicity of a representative strain of each Phytophthora species was assessed on one-year-old E. globulus seedlings as described by Bregant et al. (2023). For each isolate ten seedlings were inoculated with an agar-mycelium plug (3 mm diameter) cut from the margin of a four-day-old colony on potato dextrose agar (PDA), whereas ten seedlings used as controls were inoculated with a sterile PDA plug. Seedlings were kept in controlled conditions at 21°C and watered regularly for 30 days. All seedlings inoculated with Phytophthora isolates showed inner bark necrotic lesions spreading up and down from the inoculation point (Figure 3). Average lesion size varied significantly among isolates: P. niederhauserii 28 ±3 mm (average ±SD), P. hibernalis 10 ±3 mm and P. multivora 7 ±3 mm. The control seedlings remained asymptomatic. All Phytophthora species were successfully re-isolated from all inoculated seedlings, thus fulfilling Koch's postulates. This study represents the first report of P. hibernalis, P. multivora and P. niederhauserii on E. globulus in Portugal and in Europe. Recently, two other species, Phytophthora cinnamomi and P. alticola, have been reported as root rot agents of E. globulus in several areas of Portugal (Diogo et al., 2023). The involvement of multiple Phytophthora on E. globulus plantations is of particular concern due to the invasiveness of the species detected and the potential spread of these pathogens into natural ecosystems adjacent to eucalypt plantations. This research has been financially supported by the Land Environment Resources and Health (L.E.R.H.) doctoral course (University of Padova) and project F4F-Forest For Future (CENTRO-08-5864-FSE-000031) from Programa Operacional Regional do Centro (Centro 2020), Portugal 2020 and Fundo Social Europeu. Thanks are due to the Portuguese Foundation for Science and Technology (FCT/MCTES) for the financial support to CESAM (UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020) and the PhD grants of Sandra Hilário (SFRH/BD/137394/2018) and Eduardo Batista (PD/BD/135535/2018).

Molecular Identification, Dimorphism and Virulence of C. albicans

C. albicans causes human diseases, especially in immune-compromised patients. The current study aimed to identify Candida albicans using different techniques. Dimorphism and virulence behaviour were also studied. A Candida albicans strain was firstly identified by biochemical methods using VITEK 2 Compact automated technique and chromogenically using CHROMagar differential media that differentiate between Candida spp. Based on an enzymatic reaction. Molecular identification using ITS primers was also used to confirm Candida albicans identification. Accession number of the identified C. albicans was obtained as OK104215. The enhancement of dimorphism was studied using RPMI 1640 media (Roswell Park Memorial Institute Medium), while monitoring growth at different time intervals under microscope to investigate dimorphic changes. C. albicans showed its optimum dimorphism after 36-66 hours at 37◦C. HPLC analysis for the enzyme product S-adenosylmethionine (SAM) was carried out at different time intervals. By increasing time, SAM production increased until optimum production reached after 72h of incubation on RPMI 1640. After that the production of SAM began to decrease.

Application of Trichoderma asperellum in apple trees as a growth regulator and antagonist for the control of Alternaria sp.

Chihuahua state is the main apple producer in Mexico. The present study aimed to evaluate the effectiveness of Trichoderma asperellum as a plant growth regulator and antagonist against Alternaria sp. Three treatments were used: T1 = control, T2 = 200 mL of T. asperellum per tree; T3 = 100 mL of T. asperellum per tree. Agronomic variables were evaluated including number of leaves, shoots and flowers, disease incidence, and trunk thickness. Alternaria sp. was isolated from apple leaves at the experimental site in Guerrero County, Chihuahua, Mexico, and it was grown on solid PDA medium for morphological characterization. The molecular characterization was done by PCR using primers ITS1 and ITS4 producing products of 700 bp which were sequenced, submitted to GenBank (acc. no. OQ344593) and used for further phylogenetic analysis through Bayesian inference approach. Three clades were identified and the polytome topography recovered from clade 2 indicates a high genetic similarity with A. tenuissima (100% similarity according to BLAST). The analysis of T. asperellum as growth regulator only showed significant differences in trunk thickness and displayed higher values with T3 (p≤0.05). The presence of A. tenuissima was only observed in the control, which indicated the ability of Trichoderma to control the fungus. In this study T. asperellum was not an efficient plant growth regulator, but it was a good antagonist, and hence it can be recommended to control A. tenuissima. This is the first record of A. tenuissima in apple trees in Mexico. These results indicate that T. asperellum showed no benefit as plant growth regulator when applied to apple trees of the ‘Granny Smith’ variety.