Universal Primers ITS4 Research Articles

A Comprehensive Genetic Analysis of Mycotoxin-Producing Penicillium expansum Isolated from River Water Using Molecular Profiling, DNA Barcoding, and Secondary Structure Prediction

This study marks the first report on the genetic characterization of Penicillium expansum strain capable of mycotoxin production isolated from river water. Situated in Ganagalawanipeta village, Srikakulam, Andhra Pradesh, India, where river water serves as a vital resource, our investigation probed the presence of pathogenic opportunistic fungi adept at mycotoxin synthesis. Over six months, 30 samples were collected to assess their occurrence. This article revolves around the use of morphological traits for Penicillium genus identification. Precise species determination involved PCR analysis using universal primers ITS1 and ITS4, followed by sequence analysis through NCBI-BLASTn and the ITS2 database. The analysis indicated a striking 99.49% genetic similarity to Penicillium expansum isolate MW559596 from CSIR-National Institute of Oceanography, Goa, an Indian isolate, with a resultant 600-base pair fragment. This sequence was officially cataloged as OR536221 in the NCBI GenBank database. Sequence and phylogenetic assessments were conducted to pinpoint the strain and geographical origin. Notably, the ribosomal nuclear ITS region displayed significant inter- and intra-specific divergence, manifested in DNA barcodes and secondary structures established via minimum free energy calculations. These findings provide crucial insights into the genetic diversity and potential mycotoxin production of P. expansum isolates, shedding light on the environmental repercussions and health risks associated with river water contamination from agricultural and aquaculture effluents. This pioneering research advances our understanding of mycotoxin-producing fungi in aquatic environments and underscores the imperative need for water quality monitoring in regions reliant on such water sources for their sustenance and livelihoods.

First report of postharvest pear bitter rot caused by Colletotrichum acutatum in Italy.

The fungal genus Colletotrichum includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops, leading to yield losses (Fu et al. 2019; Talhinhas & Baroncelli, 2023). C. acutatum was reported associated with black spot on fallen, immature fruit of pear (Pyrus pyrifolia) in New Zealand (Damm et al. 2012); however, to our knowledge, this species has not been reported in Italy or nowhere else. In 2022, a significant increase of anthracnose symptoms was observed on pears in Emilia-Romagna region, Italy. Symptoms, such as round brown lesions of 1 to 4 cm, appeared on more than 50% of refrigerated stored fruit. These symptoms were undetectable at the end of September 2022 and appeared after a five-month period of storage (February 2023) at 4°C (e-Xtra 1A and B). Fungal isolates were obtained from symptomatic pears after surfaces sterilization with 96% ethanol by culturing necrotic tissue pieces on Potato Dextrose Agar at 25°C in the dark (e-Xtra 1C and D). Cultures were shades of coral color, from opalescent to sunkist coral, with slight aerial mycelium becoming grey and darker with age. When observed from the reverse side, the color was pink and, with age, became coral orange to dark amaranth. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. (e-Xtra 1E) One reference isolate, named L51, was used for molecular characterization. Total genomic DNA was extracted, and the ITS region of rDNA amplified using the universal primers ITS1 and ITS4, then sequenced. The resulting sequences were 100% identical to those of C. acutatum (NR_144794.1: strain CBS 112996 ITS region; from TYPE material). Based on Damm et al. (2012), partial ACT, GAPDH, CHS and TUB2 gene sequences were also amplified and sequenced (GenBank Accession numbers: ITS: OR882016, ACT: OR882013, GAPDH: OR882011, CHS: OR882012, TUB2: OR882010), to characterize the isolates. Additionally, the multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed the species of analyzed isolates and confirmed the BLAST results, identifying the strain as C. acutatum (e-Xtra 1F). Koch's postulates were performed on 10 'Kaiser' pears. Surfaces sterilized fruits with 96% ethanol were subjected to wound inoculation with a conidial suspension (106 conidia ml-1) while 10 fruit were used as negative control and inoculated with sterile water. Following an incubation period of 8-14 days at 15-20°C, symptoms around the inoculation site resembled those initially observed, while the negative control showed no symptoms. Fungal colonies re-isolated from the lesions exhibited the same morphological characteristics as the original isolates. To our knowledge, this is the first report of pear bitter rot caused by C. acutatum in Italy and in Europe (Talhinhas & Baroncelli, 2023). Yet, bitter rot had not been recognized as a notable issue in pear cultivation. Nevertheless, given that pears rank as the 8th most cultivated fruit globally and economically very significant for the Emilia Romagna region in Italy the emergence of pear bitter rot caused by Colletotrichum species has the potential to evolve into a significant worldwide problem, warranting further investigation.

First Report of Powdery Mildew on Cardamine violifolia in China.

Cardamine violifolia, also called Cardamine hupingshanensis, is an economically important medicinal plant renowned for accumulating selenium (Guo et al., 2022). Selenium is an essential trace element with anti-oxidant, anti-inflammatory, anti-cancer, and immune regulatory functions. In July 2023, an outbreak of powdery mildew was detected, infecting the leaves of numerous C. violifolia plants in Enshi (30°11'5.27''N; 109°48'48.45''E), Hubei Province, China. This disease caused severe damage to plant leaves and stems, starting as individual spots and merging into a large mold that covers the entire leaf. It affected nearly 25% all C. violifolia plants, resulting in significant yield loss, disruption of normal metabolism, and premature aging. The lower leaf blades and underside of the leaves were particularly vulnerable. The affected leaves were collected and subjected to morphological diagnostic analysis (Mori et al., 2000) (Fig. S1). The powdery mildew species aggressively spread throughout the leaves, pedicels, and pods, persisting until present and often covering the entire surface. The conidiophores were upright, cylindrical, composed of 3 to 4 cells, and measured 92.3 ± 12.9 × 9.2 ± 0.6 μm (n = 30). Conidial pedicels had 21.6 ± 3.4 μm (n = 50) long cylindrical podocytes. The monoconidia were columnar or barrel-columnar, 30.60-55.59 × 9.11-20.00 μm in size. Conidia lacked an obvious cellulose body. The bud tubes formed from the end of conidia, and papillary appressoria developed on the epiphytic mycelia. ITS region sequences were amplified using the specific powdery mildew universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), PM6 (5'-GYCRCYCTGTCGCGAG-3') for partial sequences of 18S and 28S ribosomal DNA genes (Takamatsu et al., 2001). The sequence was deposited in the GenBank under the accession number OR506156 and aligned with available sequences on NCBI, which were 99.2%(528/532) identical to the E. cruciferarum (MT309701, MF192845, and KY660929) sequences (Fig. S2). The ITS sequence from GenBank was used to conduct maximum likelihood phylogenetic analysis using MEGA 11.0. The analysis results showed both the strain and E. cruciferarum clustered on the same branch. To confirm Koch's postulates, pathogenicity testing was carried out using an illuminating incubator. Infected leaves were attached to healthy leaves of C. violifolia seedlings (n=8). All the plants were incubated under 25℃ and >80% relative humidity. After one month, all inoculated plants presented the same symptoms as those initially observed in the field. Morphological and molecular analysis confirmed the isolated fungi's identity as the same pathogen. Therefore, C. violifolia is a suitable host for E. cruciferarum in China. The growers must be informed of these findings to prevent serious economic losses caused by this pathogenic white powder and to prepare for proper management practices. To our knowledge, this is the first report of E. cruciferarum infecting C. violifolia in China.

Elucidating Mycotoxin-Producing Aspergillus Species in River Water: An Advanced Molecular Diagnostic Study for the Assessment of Ecological Health and Contamination Risk

The primary goal of this research is to isolate mycotoxin-producing fungus from the Nagavali River. Examining isolated fungi involved analyzing their mycelium growth on culture media and detailed microscopic inspection. We employed PCR analysis utilizing universal primers ITS1 and ITS4 to accurately identify the species. Furthermore, we sequenced the amplified ITS region and rigorously analyzed the sequences using NCBI-BLASTn and the ITS2 database. The analysis found a high 96.38% genetic similarity to the Aspergillus flavus strain, resulting in a 600-base pair fragment size. The sequence was given the accession number OR536222 in the NCBI GenBank database. Phylogenetic analysis was performed to ascertain the particular strain of A. flavus and its source. Remarkably, this analysis led to the identification of a single new strain gene, which represents a novel discovery in the field of fungal research. These results underscore the vital significance of molecular techniques in promptly and precisely identifying organisms. This research enhances our understanding of mycotoxin contamination in water, providing valuable insights to improve detection and prevention strategies. It accentuates the overarching importance of conserving our water resources and upholding ecological equilibrium, ultimately safeguarding the well-being of both humanity and the environment.

Isolation, screening and characterization of efficient cellulose-degrading fungal and bacterial strains and preparation of their consortium under in vitro studies.

In this investigation, cellulose-degrading fungi and bacteria were isolated from different partially decomposed cellulose-rich substrates, such as groundnut residues, rice straw, and rotten wood, following dilution plating techniques on carboxymethyl cellulose agar media and screening for potential cellulose degradation ability. The development of a clear halo zone surrounding the microbial colonies during the initial screening process using the Congo red test (20 isolates) suggested cellulose hydrolysis, and the highest cellulase production activity was implied by the isolates with the largest clear zone ratio (9 isolates). Using both macroscopic and microscopic examinations, as well as standard biochemical tests outlined in Bergey's Manual of Determinative Bacteriology, the genus-level identification of fungi and bacteria was accomplished. In order to molecularly identify the 4 isolatedfungal and bacterial strains at the species level after being ultimately selected for cellulase production potential under in vitro studies, fungal and bacterial DNA was extracted and amplified by PCR using the universal primers ITS1 and ITS4 for fungi (ITS rRNA, 5.8S rRNA) and 8F and 1492R for bacterial isolates (16S rRNA). After sequencing, the PCR results were compared to other comparable sequences in GenBank (NCBI). Based on the available NCBI data, phylogenetic analysis of their ribosomal gene partial sequences revealed that DAJ2 (PP086700) shares 100% homology with Aspergillus foetidus, DTJ4 (PP086699) shares 99.74% similarity with Trichoderma atrobrunnium, DBJ6 (PP082584) shares 100% identity with Priestia megaterium, and DMB9 (PP082585) shares 99.88% homology with Micrococcus yunnanensis. The cellulolytic potential of Phanerochaete chrysosporium is well established. Therefore, it was considered a standard culture for comparison and was collected from the MTCC, Chandigarh, India. Overall, all 4 selected isolates and the check organism were mutually compatible or synergistic with each other, and their consortium is useful for the accelerated decomposition of organic constituents during rapid composting.

Epidemiological etiology of Erysiphe sp. and putative viral and phytoplasma-like symptoms in Ayocote bean (Phaseolus coccineus)

Introduction/Objective. Ayocote bean (Phaseolus coccineus) has potential as a source of resistance in breeding programs because it exhibits greater tolerance to plant pathogens than P. vulgaris. However, its sanitary characterization is insipient; therefore, the purpose of this work was to carry out an etiological-epidemiological diagnosis, with emphasis on presumptive symptoms of viral and phytoplasmic organisms, and a typical fungal signs of powdery mildew. Materials and Methods. A plot (50 x 62 m) of flowering Ayocote bean was selected. It was divided into 80 (8 x 10) quadrats (6 x 6 m) and 720 subquadrats (2 x 2 m). From 25 plants with powdery-mildew-type leaf symptoms, mycelium was collected with adhesive tape for light microscopy observation and taxonomic identification. Length-width measurements were made on 60 conidia. Pure mycelium collected in situ and ex situ from 1-5 leaflets/plant was used for genomic analysis by PCR with universal primers ITS1 and ITS4. Samples were sequenced in Macrogen Inc. Korea. A total of 63 plants and 121 trifoliate leaves with viral and phytoplasmic symptoms were collected by direct sampling. In 88/121 samples, genomic analysis was performed by PCR with universal primers for Potyvirus (1), Begomovirus (2), and Phytoplasmas (1). Sequence editing and analysis were performed in SeqAssem and BLASTn/GenBank. Phylogenetic constructions were developed in Mega 11 with MUSCLE, Maximum Likelihood (ML), and HKY substitution model (1000-Bootstrap). Putative powdery mildew severity (%), flower damage (%), Macrodactylus sp. adult density, and plant vigor (%) were evaluated in 80 quadrats (3subquadrats/quadrat) with App-Monitor®v1.1 configured with a 5-class scale. In GoldenSurfer® v10, Kriging geostatistical analysis was performed to determine the spatial interrelationship between these variables. Results. Erysiphe vignae was identified as associated with powdery mildew of P. coccineus. The fungus, with hyaline, ovoid to ellipsoid conidia measuring 31.74 ± 0.3419 μm x 15.11 ± 0.1579 μm, without the presence of fibrosin bodies, had 100% genomic homology. This is the first report in Mexico. With average July-August temperature and relative humidity of 16.3 °C (±5.8) and 92.8 % (±10.7), respectively, powdery mildew leaf incidence and severity were 65.3 and 22.7 % (±16.9, range: 0 - 66.5 %), respectively. The most inductive focus (60-80 % severity) had an aggregate e 4-quadrat pattern (96 m2, lag = 4 and σ2-s = 450). Inoculum dispersal was significantly associated with dominant North-South winds and plant vigor (lag = 4 and σ2-s = 470). Flower damage was inconclusive in its spatial association with powdery mildew and Macrodactylus sp. suggesting uncorrelated events. No Potyvirus, Begomovirus, or Phytoplasmas were detected associated with yellowing, leaf distortion, mosaic, internode shortening, and other symptoms observed in situ. This confirms the relative tolerance/resistance reported for P. coccineus. Conclusion. E. vignae (Erysiphales: Erysiphaceae) associated with P. coccineus is reported for the first time in Mexico with moderate to intense epidemic level, which indicates its susceptible condition to this fungus. However, negative results for Potyvirus, Begomovirus, and Phytoplasmas, validate the apparent tolerance/resistance of P. coccineus to these organisms.

MOLECULAR CHARACTERIZATION OF MYCORRHIZA AND ITS POTENTIAL AS BIOCONTROL

Orchid mycorrhizal fungi (OMF) are vital biocontrol agents, especially for Odontoglossum ringspot virus (ORSV). The promising study helped identify the mycorrhiza isolate from native tropical orchids and determine its potential as a biocontrol. Sample collection of healthy roots of Phalaenopsis amabilis emanated in Yogyakarta, Indonesia, carrying out molecular identification with rDNA-ITS amplification using a set of universal primers ITS1 and ITS4. In vivo, antagonist tests began by inoculating viruses and mycorrhiza to determine the effect of growth and induction of secondary metabolites. The result showed one isolate of Trichoderma sp. associated with the molecular analysis has amplified the ITS1- 5.8S-ITS4 section by 600–750 bp DNA. The sequenced products revealed insertion and substitution occurrences, which may have caused the variance by strain diversity and potential severity. Indonesian isolates have undergone speciation and separation from other isolates by a substantial distance. The considerable effects were the increase in leaf length, leaf width, root length, leaf count, the number of roots, fresh weight, and a lowering of the virus content. The analysis of the plant growth parameters and virus concentrations provided significant differences among the treatments inoculated with orchid mycorrhiza (Mycorrhiza [M], Mycorrhiza + Virus [MV], and Virus + Mycorrhiza [VM]) and those without orchid mycorrhiza inoculation (Control [C] and Virus [V]). The orchid resistance suggested that the virus infecting plant leaves contain more phenolic chemicals. This study is the first-ever report of the Trichoderma sp. isolated from native tropical orchids in Indonesia.

Türkiye’nin kuzeybatısındaki bir plantasyonda yerli ve egzotik çam türlerinin ibrelerinde görülen fungal endofitler

The endophytic fungi present in needles of Pinus sylvestris, Pinus pinaster, Pinus nigra, Pinus taeda and Pinus radiata were investigated in Kerpe Research Forest, İzmit in 2016. Ten trees of each pine species were sampled systematically. Previous years green needles were sampled from the lower part of the canopy, from two equally spaced positions around the tree. Each needle was surface sterilized and cut into 0.5 cm sections then individually placed onto malt extract agar plates. DNA was ex-tracted from reprensentative seven isolates and amplified using primers ITS1 and ITS4 targeting the nuclear 5.8S rDNA gene and the two ITS regions flanked between 18S and 28S rDNA genes. Amplicons sequenced in both directions using the univer-sal fungal primers ITS1 and ITS4. Isolations from a total of 1000 needles (200 from each pine species) yielded 750 fungal isolates. Pestalotiopsis funerea (Desm.) Steyaert was isolated at the highest frequency followed by Acremonium sp., Cladio-sporum sp. and Cyclaneusma minus.

Morphological and Molecular Characterization of Major Postharvest Diseases of Mango

Mango (Mangifera indica), which is rich in nutritive value is affected by many biological factors including diseases. Postharvest diseases viz., anthracnose caused by Colletotrichum gloeosporioides and stem end rot caused by Lasiodiplodia theobromae are of economic importance due to loss in quality and quantity after harvesting. The pathogens C. gloeosporioides and L. theobromae were isolated from five mango varieties collected from fruit markets and farmers’ field of major mango growing districts of Tamil Nadu. The mycelial, colony and conidial characters were identified morphologically. Molecular characterization was carried through polymerase chain reaction using universal primers ITS1 and ITS4. The sequencing was done and the sequences were deposited in Genbank. From phylogenetic analysis Colletotrichum gloeosporioides showed maximum identity of 100% and Lasiodiplodia theobromae showed maximum identity of 88%. The present study confirmed that pathogen associated with anthracnose and stem end rot was Colletotrichum gloeosporioides and Lasiodiplodia theobromae respectively.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp (Cannabis sativa) in Missouri.

Charcoal rot, caused by Macrophomina phaseolina, is abundantly present in the soil and has been reported as pathogenic to both soybean and corn, as well as numerous other hosts, including hemp grown for fiber, grain, and cannabinoids (Casano et al. 2018; Su et al. 2001). Hemp (Cannabis sativa) production in Missouri was a relatively new addition to the 2021 growing season. Charcoal rot was reported in Reynolds, Knox, and Boone counties in Missouri from commercial and experimental fields. One of the fields in question experienced heavy disease pressure and had an uneven stand loss, but the total loss was estimated at approximately 60% of the field and was attributed to charcoal rot. Charcoal rot signs and symptoms, microsclerotia on the lower stem and root tissue, wilting and stem discoloration, were observed on a majority of the hemp plants received at the University of Missouri Plant Diagnostic Clinic in July and late Fall of 2021, including samples from Bradford Research Farm in Boone County and Greenley Research Center in Knox County. Root and crown tissue from the hemp plants from the Greenley Research Center were cultured onto acidified potato dextrose agar (APDA). Macrophomina phaseolina and other fungi grew from the plated tissue after about three days of incubation at room temperature. Macrophomina phaseolina was confirmed based on the presence of melanized hyphae and microsclerotia (Siddique et al. 2021). The microsclerotia were black, round to ovoid shaped and ranged from about 34-87 µm (average 64 µm) in length and 32-134 µm (average 65 µm) in width (n = 44). A single-hyphae isolation from a putative M. phaseolina isolate was conducted to obtain a pure culture. The M. phaseolina culture from the Greenley Research Center was used to complete Koch's postulates of charcoal rot on four hemp cultivars. Sterilized toothpicks were added to pure cultures of M. phaseolina on APDA and incubated at room temperature for one week to allow for colonization and for use in greenhouse inoculation. Four hemp cultivars (Katani, Grandi, CFX-2, and CRS-1) were grown in a sterilized silt loam for three weeks in a greenhouse. About four plants per cultivar were grown for inoculation and one plant per cultivar was used as a control. The plants were inoculated with the M. phaseolina colonized toothpicks that were gently rubbed onto stem tissue and subsequently inserted into the soil at the stem. For six weeks, the plants were kept in greenhouse conditions of 25°C with a 12-hour light and dark cycle and were watered when soil appeared dry. Plants were kept in a loosely sealed container constructed from wood and vinyl sheeting to minimize cross contamination with other plants grown in the same greenhouse. Plants were monitored weekly for charcoal rot symptoms. Symptoms that resembled charcoal rot, wilting and microsclerotia on the lower stem, were present on inoculated plants after about four weeks and symptoms were not present on the control plants. Isolates resembling M. phaseolina in culture were recovered from symptomatic plants; therefore, Koch's postulates were successfully fulfilled and the fungus was recovered from the inoculated plants. DNA was extracted from the pure cultures of both the initial isolate and the isolate obtained from Koch's postulates using GeneJet Plant Genomic DNA Purification Kit (Thermo Scientific, California, USA) and the internal transcribed spacer (ITS) region of ribosomal DNA including ITS1, 5.8S, and ITS4 regions were amplified using universal primers ITS1 and ITS4 (White et al. 1990). The ITS region was sequenced and compared to reference sequences in GenBank by BLAST analysis. Recovered isolates (GenBank accession no. OQ455934.1) showed closest sequence similarity (100%) to M. phaseolina accession number GU046909.1. Little is known about the life cycle, growth conditions, and possible inoculum buildup in the soil in hemp in Missouri. In addition, M. phaseolina is a known pathogen of corn and soybean and effective management strategies are challenging for these crops as well due to the broad host range of the pathogen. Cultural management practices, such as crop rotations to reduce inoculum in the soil and closely monitoring for symptoms, may help reduce the severity of this disease.

Morphological and Molecular Characterization of Phytophthora palmivora Isolates and Their Virulence Test on Cocoa Clone Sulawesi 2

Morphological characterization of Phytophthora isolates commonly was done on the basis of the colony and hyphal swelling characteristics, form, and shape of chlamydospore, sporangia as well as oogonia and antheridia. Molecular characterization was conducted using specific primer pairs Pal1 s and Pal2a for Phytophthora palmivora and universal primer ITS4 and ITS5 followed by sequencing of isolates. The virulence of Phytophthora palmivora isolates was tested on a cocoa pod of the Sulawesi2 clone. A total of 21 Phytophthora isolates were collected from Palopo, Gowa, and Pinrang Districts. Based on colony morphology and microscopic features i.e. chlamydospore, sporangium, and its papilla form, all isolates indicated Phytophthora palmivora. Molecular identification using a specific primer showed that all ten representative group isolates had the same band in size of 650 bp, whereas using universal ITS4 and ITS5 primers, all isolates generated one band in the size of 900bp. The sequencing result showed, that all isolates were placed in the group P. palmivora. The virulence of P. palmivora isolates on the Sulawesi2 clone revealed a varying degree of virulence, from less virulent to highly virulent. All isolates from Pinrang were classified as highly virulent because induced necrotic in more than 50% of the tested cocoa pod.

First Report of Pseudoidium santalacearum Causing Foliar Powdery Mildew on Santalum album in Guangdong Province, China.

Santalum album Linn is an evergreen and facultative root hemiparasitic tree. It is attached to host roots through haustorium to withdraw water and nutrients. Its aromatic heartwood, honored as "Green Gold", is widely used in perfumes, incense, medicine, and skin cancer prevention (Sandeep and Manohara 2019, Polaiah et al. 2020). From February to June 2022, powdery mildew was observed on over 80% of S. album in the nursery with a mean temperature and relative humidity of 30/22°C (day/night) and 55/82% (day/night) at the Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangdong, China (23°11'N, 113°23'E). Initial symptoms were circular to irregular white colonies on the leaf surface (Fig. 1). As the disease progressed, white mycelia covered the entire leaf surface and premature defoliation of the infected leaf was observed. Then a bare stem remained and subsequently desiccated (Sundararaj et al. 2022). Infected leaves with mycelia were collected and observed under a Zeiss AX10 microscope. Foot cells of conidiophores (n = 30) were straight to slightly curved cylindrical, and the length and width of foot cells were up to 24.8 to 44.6 × 8.2 to 11.7 μm. Conidiophores (n = 30) were straight, 83.5 to 127.3 μm long × 6.6 to 7.6 μm wide and produced three immature conidia in chains. Conidia (n = 30) were elliptical to cylindrical, measured 31.3 to 41.2 μm × 11.3 to 13.7 μm. The sexual stage was not found. The morphological characterization of our samples was consistent with powdery mildew of S. album in India, termed Pseudoidium santalacearum (Patel et al. 2015). However, the molecular identification was not performed. In this study, we carefully collected the mildew and extracted the genomic DNA. The internal transcribed spacer (ITS) region of rDNA was amplified using universal primers ITS1 and ITS4 (White et al. 1990). The ITS sequences of the three samples (GenBank Accession No. OQ746371, OQ746372, and OQ746373) were all more than 99% identical with the sequence of Erysiphe palczewskii on Caragana arborescens (GenBank Accession No. LC010048) (Takamatsu et al. 2015). It is known that Erysiphe containing chasmothecia belongs to teleomorph, whereas Pseudoidium is an asexual morph of Erysiphe, belonging to anamorph (Hsiao et al. 2022, Meeboon and Takamatsu 2016). In our samples, there was no chasmothecia, and the morphological characterization was consistent with P. santalacearum (Patel et al. 2015). Therefore, we classified it as P. santalacearum. Phylogenetic analysis showed that P. santalacearum had a closer relationship with the E. palczewskii (Fig. 2). Pathogenicity tests were conducted by gently pressing the infected leaves onto five healthy S. album plants. Five non-inoculated plants were used as controls. The plants were placed in a growth chamber (Light/Dark: 16 h/8 h, temperature: 25 ± 2°C). Inoculated leaves developed powdery mildew symptoms after 7-12 days, whereas the control plants remained symptomless (Fig. 1). Morphological and molecular characters of the fungus on the inoculated plants were identical to those on naturally infected S. album, fulfilling Koch's postulates. To our knowledge, this is the first report of P. santalacearum on S. album in Guangdong, China. Powdery mildew can generally result in a high mortality of S. album seedlings. Therefore, identification of the pathogen provides a scientific basis for effective diagnosis and prevention of the disease and contribute to an appropriate nursery management of S. album seedlings.

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.

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).

Comparison of in vitro activities of newer triazoles and classic antifungal agents against dermatophyte species isolated from Iranian University Hospitals: a multi-central study

BackgroundDermatophytes have the ability to invade the keratin layer of humans and cause infections. The aims of this study were the accurate identification of dermatophytes by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method and sequencing and comparison between the in vitro activities of newer and established antifungal agents against them.MethodsClinical specimens of patients from five Iranian university laboratories were entered in this study. Samples were cultured on sabouraud dextrose agar medium. For molecular identification, extracted DNAs were amplified by the universal fungal primers ITS1 and ITS4, and digested with MvaI enzymes. The antifungal susceptibility test for each isolate to terbinafine, griseofulvin, caspofungin, fluconazole, itraconazole, luliconazole, and isavuconazole was performed, according to the microdilution CLSI M38-A2 and CLSI M61 standard methods.ResultsTwo hundred and seven fungi species similar to dermatophytes were isolated of which 198 (95.6%) were dermatophytes by molecular assay. The most commonly isolated were Trichophyton mentagrophytes (76/198), followed by Trichophyton interdigitale (57/198), Trichophyton rubrum (34/198), Trichophyton tonsurans (12/198), Microsporum canis (10/198), Trichophyton simii (3/198), Epidermophyton floccosum (3/198), Trichophyton violaceum (2/198), and Trichophyton benhamiae (1/198). The GM MIC and MIC90 values for all the isolates were as follows: terbinafine (0.091 and 1 μg/ml), griseofulvin (1.01 and 4 μg/ml), caspofungin (0.06 and 4 μg/ml), fluconazole (16.52 and 32 μg/ml), itraconazole (0.861 and 8 μg/ml), isavuconazole (0.074 and 2 μg/ml), and luliconazole (0.018 and 0.25 μg/ml).ConclusionTrichophyton mentagrophytes, Trichophyton interdigitale, and Trichophyton rubrum were the most common fungal species isolated from the patients. luliconazole, terbinafine, and isavuconazole in vitro were revealed to be the most effective antifungal agents against all dermatophyte isolates.

Alternaria alternata Causing Brown Spot Disease on Walnut in Xinjiang, China.

Walnut is one of the Xinjiang's characteristic dried fruits and the main source of income for farmers in walnut growing areas. In September 2019, Juglans regia leaves with brown spots were observed in a 10 hm2 orchard in Hotan area, the diseased leaf rate reached more than 25%. The leaf lesions were suborbicular to irregular, black-brown, 3 to 8 mm in diameter, with distinct dark borders. Colonies were isolated from 10 diseased leaves collected from two trees in the orchard. Leaf sections (4 × 4 mm) from diseased leaves were surface disinfested with 75% ethyl alcohol for 30 s and 2% NaClO for 3 min, washed with sterile water three times and then plated on potato dextrose agar (PDA) and incubated at 27℃ with a 12h/12h light/dark photoperiod for 4 days. A total of 7 fungal isolates were obtained by single-spore isolation. All the colonies were dark olivaceous on the PDA plates, with loose, cottony mycelium. On potato carrot agar (PCA), all fungal isolates produced conidial chains with numerous secondary chains. The conidia were ellipsoid or obpyriform with 0-3 longitudinal septa and 2-4 transverse septa, measuring 20.6 to 35.8 × 6.8 to 11.2 μm (25.5 ± 0.4 × 8.7 ± 0.2 μm, n=50). The morphological characteristics of the seven fungal isolates were consistent with the A. alternata descriptions of Simmons (2007). DNA was extracted from 50 mg of mycelia for the representative isolate HLP17-7. The internal transcribed spacer (ITS) region was PCR amplified using the universal primers ITS1 / ITS4 (White et al.1990), the partial coding sequence of endopolygalacturonase (endoPG) and the partial region of the histone 3 (H3) were amplified using primers PG2b / PG3 (Andrew et al. 2009) and H3-1a / H3-1b (Glass and Donaldson 1995) respectively. The products were sequenced and deposited in GenBank database under the accession numbers MW514319 [ITS], ON806938 endoPG, MW489301 [H3]. ITS, endoPG and H3 sequences had 99.81% (1/535 nt difference), 99.78% (1/448 nt difference) and 100% (0/417 nt difference) homology with homologous sequences of A. alternata strains (KP124306 [ITS], KP124006 [endoPG], MK085979 [H3]), respectively. During the early autumn, pathogenicity tests were carried out on the healthy mature leaves of seven-year-old Juglans regia plants in the field. Thirty leaves (five leaves per plant) were wounded with a sterile needle and then sprayed with a spore suspension prepared from 10-day-old PDA culture. Five wounded leaves per plant were sprayed with sterile water as control. All the treated leaves were covered with clear plastic bags for 3 days, and the experiment was replicated three times. On the 8th day after inoculation, brown spots appeared on the inoculated leaves, but no spots were observed in the control. Morphological observation and gene sequencing confirmed that the original fungal pathogen was re-isolated from the inoculated leaves. No colony was isolated from the control leaves. The pathogen causing the brown spot was identified as A. alternata based on morphological features and sequence analysis. A. alternata has been reported previously in Sichuan (Yang et al., 2017) causing brown spot in walnut. Xinjiang is dry with little rain and abundant sunshine, so there are few diseases on walnuts. However, the occurrence of brown spot disease has alarmed fruit farmers, walnuts are still at the risk of A. alternata infections even in dry environment with little rain. To our knowledge, this is the first report of A. alternata causing brown spot in walnut in Xinjiang, China.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Glandularia tenera in China.

Glandularia tenera (syn. Verbena tenera) is an herbaceous perennial ornamental plant used in gardens as an edging plant with beautiful white, red, or purple flowers. In autumn 2020 and 2021, severe powdery mildew infection was observed on G. tenera cultivar Xianghe in Renming Botanical garden in Shangqiu, Henan province, China (34.4568° N, 115.6640° E). Approximately 80% of leaves on each plant were symptomatic, and about 90% of the plants were infected. Powdery mildew colonies appeared as white spots on the adaxial surface of the leaves and stems of the plants in the initial infection stage. Later, mycelial growth was amphigenous, thick, forming irregular white patches, and effused to cover the whole leaf surface. Finally, leaves turned yellow and senescence occurred. Samples of symptomatic leaves were stained with trypan blue and examined under a Leica DM2500 microscope. Microscopic observations showed that conidia on infected leaves were hyaline and ellipsoid to oval with fibrosin bodies, measured 25 to 37 × 14 to 23 μm with a length/width ratio of 1.4 to 2.0. Conidiophores were unbranched, straight, 80 to 210× 10 to 14 µm in size, and produced two to five immature conidia in chains. Foot cells of conidiophores were cylindrical with slight constrictions at basal septa, and followed by one to three short cells. Fungal hyphae were septate, branched, and flexuous to straight and 4 to 7µm wide with indistinct to slightly nipple-shaped appressoria. Chasmothecia were not observed. These morphological characteristics were identical with the previous description of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012). To confirm the identification, the sequence of ITS1-5.8s-ITS2 region of rDNA for the isolate SQVT was amplified from conidia collected from infected leaves with universal primers ITS1 and ITS4, sequenced and analyzed using the BLASTn search of GenBank. Amplicon was 565 bp (OM293967) and showed 99.82% similarity with sequence of P. xanthii from Eclipta prostrate (MT260063) in China (Xu et al. 2020), from Youngia denticulate (AB040351) in Japan (Hirata et al. 2000), and 99.65% with sequence of P. xanthii from V. brasiliensis in Korea (Cho et al. 2014). The domains D1 and D2 of the 28S rDNA for the isolate SQVTPX-1 was amplified with primer NL1/NL4. Amplicon was 613 bp (ON259308) and showed 100% similarity with sequence of P. xanthii from V. brasiliensis (AB936277) (Meeboon and Takamatsu, 2015). Pathogenicity tests were conducted by gently pressing the infected leaves onto leaves of five healthy G. tenera cultivar Xianghe plants. Five non-inoculated plants served as controls. Plants were maintained in a greenhouse at 25 ± 2°C. Eight days after inoculation, symptoms similar to those observed under natural conditions developed on the inoculated leaves of G. tenera plants, whereas the control plants remained symptomless. The fungus on inoculated leaves was morphologically identical to that first observed in the field. P. xanthii is a cosmopolitan powdery mildew fungus, parasitic on numerous plant species, especially Cucurbitaceae and Compositae plants. The pathogen has been reported infecting V. bonariensis (Hong et al. 2021), V. × hybrida in China (Zhuang 2005), and V. brasiliensis in Korea (Cho et al. 2014). Interestingly, G. tenera plants infected by P. xanthii were adjacent with V. × hybrida plants infected by P. xanthii in Renming Botanical garden. Incidence of P. xanthii on G. tenera add information on pathogen's host range and help us develop comprehensive survey and effective management of the disease. To our knowledge, this is the first report of P. xanthii on G. tenera in China (Farr and Rossman 2021). Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series No. 11. CBS, Utrecht, the Netherlands. Cho, S. E., et al. 2014. Plant Dis. 98: 1159. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Hong, Q. Q., et al. 2021. Plant Dis. 105: 3297. Hirata, T., et al. 2000. Can. J. Bot. 78: 1521-1530. Meeboon, J. and Takamatsu, S. 2015. Mycoscience . 56: 243-251. Xu, D. D., et al. 2020. Plant Dis. 104: 3263. Zhuang, W. Y. 2005. Fungi of northwestern China. Mycotaxon, Ltd., Ithaca, NY.

First Report of Phytophthora inundata Associated with Decline and Death of Walnut (Juglans regia L.) in Italy.

Persian walnut (Juglans regia L.) is an important nut crop in Italy. In recent years, incidence of walnut decline and death has increased in many Italian commercial orchards. In early summer 2020, we observed a serious decline in approximately 5% of trees in a waterlogged area of a Veneto-region walnut orchard (J. regia, cv Lara). Symptoms included extensive foliar wilt and canopy decline associated with collar and root rot. Symptomatic tissues excised from larger roots of affected trees were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in sterile distilled water, and placed onto P5ARPH selective medium (Jeffers and Martin 1986). A Phytophthora-like organism was consistently isolated. Pure cultures were obtained by single-hyphal transfers onto potato dextrose agar (PDA). Isolates were identified as Phytophthora inundata based on morphological characteristics (Brasier et al. 2003), sequences of internal transcribed spacer (ITS) amplicons from universal primers ITS6 (Cooke et al. 2000) and ITS4 (White et al. 1990) and sequences of cytochrome c oxidase, subunit II (Cox II) from Fm75 and Fm78 primer pair (Martin and Tooley 2003). On carrot agar (CA), colonies had a characteristic stellate to broad lobed patterns. On this medium, optimal growth was at 28-30 °C (7,3 mm/day) and the upper temperature limit for mycelial growth was 37°C. Mycelial disks of isolate CREADC-Om306, grown on CA, were floated in Petri plates with soil extract solution and incubated under continuous fluorescent light at room temperature (25+/-2 °C). Within 48 to 72 h, sporangia were produced that were persistent, non-papillate, ovoid or ovoid-obpyriform, measuring 55.0 to 80.7 (length) x 41.3 to 65.2 (width) µm (averages 64.3+/-10.2 x 47.9+/-9.7 µm). Oospores and chlamydospores were absent. BLAST analysis of the amplicons from CREADC-Om306 revealed ITS sequences (854-bp; GenBank accession no. OK342200) and Cox II sequences (568-bp; GenBank accession no. OK349677) that shared 100% identity with published P. inundata sequences available in GenBank (acc. n. AF266791 for ITS; MT458994 for Cox II). Pathogenicity tests were conducted in the greenhouse on six 2-year-old walnut (J. regia, cv Lara) plants. Four of the plants were inoculated with CREADC-Om306 on two opposite sides of each plant's stem at 1-2 cm above soil line. A cork borer was used to remove a 5-mm disk of bark that was replaced by a 5-mm diameter mycelial plug from 10-day-old cultures of the pathogen on PDA. Two control plants were treated in the same way except the bark wounds were inoculated with sterile PDA plugs. Plants were kept in greenhouse at 24 ± 2°C. After 3 months, lesions had developed from all points of inoculation with. P. inundata (mean lesion length 55,25+/-6,22 mm) and the pathogen was reisolated from the lesion margins of all inoculated plants. The control plants remained symptomless and did not yield the pathogen. P. inundata is widely distributed across the world as a plant pathogen on several native as well as horticultural crops, especially in riparian or other areas subject to flooding or waterlogging. This report is the first to document P. inundata as a pathogen on Persian walnut and adds it to the diverse list of known susceptible perennial native, ornamental, and agricultural hosts of this organism. In addition to P. inundata, which belongs to the major Phytophthora ITS Clade 6, other members of the clade including P. megasperma (Belisario et al. 2012) and P. gonapodyides (Belisario et al. 2016) have been described as walnut pathogens.

P213 Lasiodiplodia theobromae: an emerging human pathogen

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Lasiodiplodia theobromae is a dematiaceous fungus, rarely reported to cause infections in humans. This case-series was conducted to identify the potential risk-factors and spectrum of diseases caused by L. theobromae.MethodsWe performed a retrospective analysis of all cases from which the isolates of the L. theobromae were accessioned at the National culture collection of pathogenic fungi (NCCPF), over the last 10 years (January 2012- March 2022). The isolates were retrieved and identified by conventional (colony morphology, color, and microscopic appearances) and molecular (sequencing of ITS1 region of ribosomal DNA) methods. In vitro antifungal susceptibility testing (AFST) was performed by microbroth dilution recommended by the Clinical and Laboratory Standards Institute (CLSI). (M38-A2). The clinical features, demographic details and outcome were recorded.ResultsIn 10 years, a total of 20 patients were identified. The median age of all cases was 39.5 (range: 13-71 years) and 75% were males. The most common clinical presentation was keratomycosis (12,68.6%), followed by soft tissue/sub-cutaneous infection (3,15%), rhinosinusitis (3,15%), onychomycosis (1, 5%), and pneumonia (1, 5%). Most patients were immunocompetent (85%). Among immunocompromised, two patients had acute myeloid leukemia and developed fungal rhinosinusitis while one patient of post-renal transplant on immunosuppressants developed subcutaneous tissue infection in the interscapular region. The fourth patient had decompensated alcoholic hepatitis, encephalopathy, sepsis, and developed pneumonia while he was on mechanical ventilation. Among the various potential risk factors associated with keratomycosis, the most common were, accidental trauma wounds (9, 45%), and mosquito bite in the eye in one patient. The culture on Sabouraud's dextrose agar (SDA) revealed the growth of black mycelial fungus (Fig. 1a). The lactophenol cotton blue (LCB) mount revealed the presence of only dematiaceous septate hyphae which failed to sporulate (Fig. 1b). All the isolates were confirmed by sequencing of ITS (internal transcribed spacer) region of the rDNA using universal primers ITS1 and ITS2. In vitro antifungal susceptibility testing (AFST) was performed using a broth microdilution (BMD) method which revealed variable MIC (μg/ml), i.e., amphotericin (1-2), voriconazole (0.25-2), itraconazole (8-16), posaconazole (1-2). All patients improved on therapy except one patient who succumbed to death due to pneumonia.Conclusion Lasiodiplodia theobromae is an emerging cause of human infections in both immunocompetent and immunosuppressed individuals. It is often difficult to identify due to lack of sporulation making morphological identification challenging. Hence, prompt suspicion and rapid diagnosis with guided therapy are necessary for a better outcome.

Morphological and Molecular Characterization of Colletotrichum gloeosporioides Causing Mango Anthracnose

Mango (Mangifera indica), a fruit with high nutritional value is widely regarded as a most palatable fruit crop which is affected by a number of biological constraints mainly diseases. Anthracnose caused by Colletotrichum gloeosporioides belongs to order Melanconiales is the important post harvest disease which restricts the marketing of mango. In the present study, thirteen isolates obtained from various places of Tamil Nadu were collected and isolated by tissue segment approach and purified. The isolates were confirmed phenotypically using morphological characters. Molecular investigation like PCR assay using universal primers ITS1 and ITS4 produced amplicon size of 560bp. The isolates were also identified using genus specific and species specific primers which resulted in the amplicon size of 280bp and 380bp. As a result, The use of morphological and molecular approaches to characterise Mango anthracnose will be useful in identifying and managing the disease.