Internal Transcribed Spacer Gene Research Articles

Occurrence of Neopestalotiopsis clavispora Causing Leaf Spot on Dendrobium officinale in China.

Dendrobium officinale Kimura L., an endangered orchid plant, is a rare and precious Chinese herb and widely used to prepare Chinese traditional medicine (Zheng et al. 2005). In August 2021, significant indications of an unknown leaf spot disease were observed on greenhouse-grown D. officinale in Yueqing of Wenzhou (28.39°N, 121.04°E), Zhejiang Province, China, the main producing location of this orchid plant. Approximately twenty percent of plants surveyed showed typical infection symptoms. Initially, the symptoms appeared as small, circular black spots. As the disease developed, the center of the lesions was sunken with a black border. To determine the causal agent, 10 symptomatic plant samples were collected and all pieces from symptomatic plant leaves were used for isolating pathogen. Tissues between healthy and necrotic area were cut into pieces (5 × 5 mm, n=10), disinfected with 10% sodium hypochlorite for 1 minute, rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar medium (PDA) for 1 piece per plate, and incubated at 25℃ in a dark biochemical incubator. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. Twelve same fungal isolates were obtained from all symptomatic leave fragments, then DDO11 was chosen as a representative isolate for further study. The colonies showed white aerial mycelium after 5 days culture at 25°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 8-12 days culture. Conidia were spindle shape, five cells, four septa, average 29.3 × 8.5 μm (n = 30; length × width). The apical and basal cells were lighter in color, and most of them were hyaline. The middle three cells were darker in color, and mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 32.5 µm in average length, and the basal cell has a small appendage, 9.2 µm in average length, n=30. For fungal identification to species level, Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. The ITS, TUB2 and TEF-1α gene sequences of the representative isolate DDO11 were deposited in NCBI GenBank nucleotide database with accession numbers OK631881, OK655895 and OK655896, respectively. BLASTn analysis respectively showed 100%, 100% and 99.6% nucleotide sequence identity with Neopestalotiopsis clavispora strain accessions MG729690, MG740736 and MH423940, which indicated that the pathogen belonged N. clavispora. A maximum-likelihood phylogenetic analysis based on multi-locus sequence (ITS, TUB2, and TEF-1α) using MEGA X showed the similar result (Kumar et al. 2018). To verify pathogenicity, thirty 1-year-old healthy D. officinale plants of cultivar Yandang1 were used for inoculation tests. Spores of N. clavispora DDO11 were produced on PDA for 7 days at 28°C and washed with sterile distilled water, and the concentrations were adjusted to 1 × 106 spores/ml using a hemocytometer. Fifteen surface disinfected healthy plants were inoculated by spraying the suspension (2 ml, 1 × 106 spores/ml) and covered with plastic bags for 24 h, and another 15 plants treated with sterile distilled water were used as control. The plants were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber (Kiangnan, China) at 25°C with 12-h day/night cycle for 8 days (Cao et al. 2019). All inoculated leaves showed symptoms identical to those observed in the field. No disease occurred on the controls. The Neopestalotiopsis isolate was reisolated from the symptomatic leaves, and species identification was confirmed by the morphological and molecular method described above. N. clavispora has been reported to cause diseases on a variety of plants all over the world, such as strawberry (Gilardi et al. 2019), blue berry (Shi et al. 2021), Syzygium cumini (Banerjee et al. 2020), Macadamia (Qiu et al. 2020), and so on. To the best of our knowledge, this is the first report of N. clavispora causing leaf spot on D. officinale in China. This report will help us to recognize the leaf spot disease of D. officinale and establish a foundation for future studies on N. clavispora to address effective management strategies.

Morphological and Molecular Characterization of Some Alternaria Species Isolated from Tomato Fruits Concerning Mycotoxin Production and Polyketide Synthase Genes.

Tomatoes (Lycopersicon esculentum) are one of the main crops grown in Egypt. The fungal black spot illness of fruits is usually associated with the secretion of mycotoxin by Alternaria toxigenic species. Twenty Alternaria isolates were isolated from infected tomatoes fruits by baiting technique, morphologically identified to species level, and confirmed using Internal Transcribed Spacer (ITS) gene sequencing. ITS gene sequencing of fragments obtained 547, 547, 542, 554, and 547 bp for A. alternata, A. brassicicola, A. citri, A. radicina, and A. tenuissima, respectively. Alternaria species were investigated for mycotoxin production using the high-performance liquid chromatography (HPLC) technique. The data from the HPLC analysis showed that the mycotoxins were determined in four out of five Alternaria species, with the incidence ranging from 0.89–9.85 µg/mL of fungal extract at different retention times. Alternaria alternata was the most active species and produced three types of toxins. Polyketide synthase genes (pksH and pksJ) which are involved in the Alternaria toxin’s biosynthesis were also amplified from the DNA of Alternaria species.

Internal Transcribed Spacer (ITS) gene as an accurate DNA barcode for identification of macroscopic fungus in Aceh

Abstract. Harnelly E, Kusuma HI, Thomy Z, Samingan. 2022. Internal Transcribed Spacer (ITS) gene as an accurate DNA barcode for identification of macroscopic fungus in Aceh. Biodiversitas 23: 2369-2378. Fungal identification is crucial in determining the fungal’s role and benefits and is usually carried out by morphological observation. However, this technique takes a long time and depends on the accuracy of an expert. Therefore, the present study aimed to examine the accuracy of the Internal Transcribed Spacer (ITS) gene to identify macroscopic fungi from Aceh, Indonesia. 23 samples were collected from the Pocut Meurah Intan Grand Forest Park in Aceh, Indonesia. Samples were extracted, amplified, electrophoresed, and sequenced. The nucleotide sequence of the amplified products was analyzed by using the Basic Local Alignment Search Tool (BLAST). 7 out of the 11 samples with good and regular peaks were continued until further analyzed using BLAST. The results showed that samples aga2, aga4, aga5, aga6, aga9, rus, and pol were identified as Pleurocollybia sp., Pleurotus djamour, Filoboletus manipularis, Schyzophillum commune, Gymnopilus lepidotus,Auriscalpium vulgare, and Gyrodontium sacchari, respectively. The study findings indicate that the use of the ITS gene as a barcode effectively separates up to the species level from the type. This is very helpful in the identification process, especially when distinguishing cryptic species clearly because of their similarity, which is difficult to distinguish morphologically.

First Report of Cirsium arvense (Canada Thistle) as a New Host of Orobanche cumana in Xinjiang, China.

Cirsium arvense (Canada thistle) is a perennial herb native to Eurasia that has been introduced to temperate regions of the world where it is considered one of the serious weeds for arable and pastoral agriculture (Schröder et al. 1993). C. arvense reproduces both clonally and sexually. The weed is highly competitive, causes yield reductions in crops such as wheat, alfalfa, sugarbeet, and can reduce forage availability and production (Wilson 1981). Canada thistle is also a harbour for plant pathogens such as plant-parasitic nematodes (Tenuta et al. 2014). Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant species with a restricted range of hosts both in the wild, where it mainly parasitizes a few species of the Asteraceae, and in agricultural fields, where it is exclusively found growing on sunflower (Fernández-Martínez et al. 2015). O. cumana infection can cause up to 80% of the yield loss in sunflower, which is a serious threat for sunflower production in Xinjiang and Inner Mongolia, China (Parker 2009). In July 2019, broomrape was observed parasitizing C. arvense in the greenhouse used for sunflower resistance identification (Shihezi, 86° 3' 36" E, 44° 18' 36" N, 500 m elevation) in Xinjiang, China. Fifty percent of the plants were parasitized by broomrape in the greenhouse and the host had an average of 1-2 broomrape shoots per plant. For molecular analysis, total genomic DNA was extracted from the flowers of broomrape and the rps2, rbcL, trnL-F genes, and ribosomal DNA internal transcribed spacer (ITS) region were amplified by PCR using the primer pairs rps2F/rps2R, rbcLF/rbcLR, C/F, ITS1/ITS4, respectively (Park et al. 2007; Manen et al. 2004; Taberlet et al. 1991; Anderson et al. 2004). The ITS (659bp), rps2 (451 bp), trnL-F (914 bp), and rbcL (961 bp) gene sequences of the broomrape were deposited in GenBank, the accession numbers are MT856745, MW809407, MW809408, and MW809409. The results of BLAST analysis showed that ITS sequence shared 100% similarity with O. cumana (659/659 nucleotide identity, MK567978), the rps2 sequence shared 99% similarity with O. cumana (449/451 nucleotide identity, KT387722), trnL-F sequence shared 99% similarity with O. cumana (907/911 nucleotide identity, MT027325), rbcL sequence shared 99% similarity with O. cumana (956/964 nucleotide identity, MK577840). The morphological characteristics such as stem, inflorescence, corolla, bracts, calyx, stamens, gynoecium are consistent with O. cumana described by Pujadas-Salvá and Velasco (2000). Morphological and molecular identification strongly support that the broomrape parasitic on C. arvense belonged to the O. cumana. Greenhouse pot experiments were carried out to assess the parasitic relationship between sunflower broomrape and C. arvense (Fernández-Martínez et al. 2000). In January 2020, C. arvense roots were harvested from an extant field of C. arvense in the greenhouse at Shihezi University (Supplementary Figure S1A). The soil was dug to 30-40 cm depth and C. arvense roots were removed and carefully washed in water. The healthy and living C. arvense roots were selected and cut into 10-11 cm pieces. Four C. arvense root pieces were grown (buried at a depth of 10-12 cm) in 8-L pots containing a mixture of sand-vermiculite-compost (1:1:1 v:v:v) and O. cumana seeds (50 mg of O. cumana seeds per 1 kg of the substrate) with 5 replicates. Three non-infected plants were grown and evaluated in parallel. Approximately 80 days after planting, at the flowering stage of the O. cumana, C. arvense plants were uprooted from the soil. Compared to non-infected plants, the hosts' symptoms were slow growth, leaf wilting, and chlorosis, and similiar to the broomrape-infected C. arvense plants observed in the greenhouse field. The roots of C. arvense and broomrape were carefully washed in water and observed the parasitism of O. cumana. The infection was confirmed by observation of the attachment of the O. cumana to the C. arvense roots (Supplementary Figure S1D). To the best of our knowledge, this is the first report of O. cumana parasitizing C. arvense in Xinjiang, China. C. arvense as a new host of O. cumana indicates that sunflower broomrape can also propagate and survive in a host such as Canada thistle grown in sunflower fields. This finding suggests that it may be more difficult to control sunflower broomrape by rotation. In the next study, the contaminated area and the degree of parasitism of broomrape on C. arvense in the field will be investigated, and better-integrated control methods for controlling O. cumana will be designed.

First Report of Exserohilum rostratum Causing Leaf Spot on Maize (Zea mays) in Henan, China.

In August to September of 2019 and 2020, a leaf spot was observed on maize at growth stages R1 through R5 in Henan. Leaf samples (N = 63) were collected from 152 fields from 50 counties. Symptomatic plants had light-yellow to brown elliptic lesions (0.6 to 5.0 cm × 0.2 to 0.8 cm) with gray. Incidence rates were 1 to 15% in individual fields. Symptomatic leaf samples (3 to 5 cm2) were surface sterilized (75% ethanol, 30 s; 3% NaClO, 2 min), rinsed three times in sterile distilled water, and allowed to dry. Small sections from the samples were placed on potato dextrose agar (PDA) medium and incubated at 25°C for 4 days. Observed colonies were circular, taupe, pewter in color with light gray edging and many cottony aerial mycelia. Single spores (N = 45) were inoculated on water agar medium with wheat straw (TWA + W). Conidiophores on wheat straw were single, erect or slightly curved, in clusters, septate, sympodial, geniculate and 5 to 7 μm thick. The basal cell was slightly swollen and was dark brown. The basal septum was darker and thicker than other septa, with a distinctly protruding truncate hilum. The conidia belonged to two types (Lin et al. 2011). Type A were fusiform, cylindrical, pyriform to oval, dark brown, 2 to 7 septate, 16.5 to 70.5 µm × 10.2 to 24.7 µm (N = 50). Type C were straight or slightly bent, ellipsoidal or long ellipsoidal, light brown, 6 to 13 septate, 36.3 to 106.8 µm × 10.5 to 16.0 µm (N = 50). All single spore isolates were identified morphologically as Exserohilum rostratum (Drechs.) Leonard & Suggs (Ahmadpour et al. 2013; Sivanesan et al. 1987). Four isolates (ML154-1, ML300-2, ML598-4 and ML626-1) were arbitrarily selected for molecular identification and inoculation testing. The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase-like (gpd) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and Gpd1/Gpd2 (Berbee et al. 1999), respectively. Sequences MW440488, MW440489, MW440491 and MW440492 of ITS and MW448214, MW448215, MW448217 and MW448218 of gpd were submitted to GenBank. BLASTn analysis revealed a high match (ITS: 598/600 bp; gpd: 527/527 bp) to E. rostratum isolate BRIP11422 (ITS: LT837464; gpd: LT883541). A phylogenetic tree was constructed based on concatenated sequences of ITS and gpd gene in MEGA-X. All four isolates formed a single clade with E. rostratum reference isolates CBS732.96, BRIP11422 and BRIP11416. To test pathogenicity in the greenhouse, a conidial suspension of each of the four strains (1 × 104 conidia/ml) was sprayed on the leaves of the common maize hybrids, Zhengdan 958 and Xianyu 335 (V6- V7). Control plants were sprayed with sterile water. Plants were incubated in plastic bags to retain high relative humidity for 24 h at 25~27°C. Three days after inoculation, the leaves began to show symptoms, and 7 days later, symptoms of the leaves were identical to those observed in the field. Control plants developed no symptoms. The pathogen was re-isolated from the infected leaves and was confirmed to be E. rostratum. The first E. rostratum isolate was isolated from the leaves of Eragrostis in China (Jiang et al. 1959). Thereafter E. rostratum has been reported as the causal agent of leaf spot in Ananas comosus (He et al. 2011), Musa paradisiacal (Luo et al. 2012), Hevea brasiliensis (Zhang et al. 2016) and many gramineous grass weeds (Liu et al. 2016) in China. To our knowledge, this is the first report of E. rostratum causing leaf spot disease on maize in Henan, China. Since this is an economically important pathogen of maize throughout the world, disease management strategies are likely to be needed.

First Report of Fusarium oxysporum f. sp. ciceris on Chickpea (Cicer arietinum) in Serbia

Chickpea (Cicer arietinum L.) is one of the most commonly consumed legume crops worldwide, cultivated in more than 55 countries (FAOSTAT, 2017). However, in Serbia, it is a novel crop grown on approximately 120 acres, but the areas under this crop slightly increase each year. Fusarium wilt caused by F. oxysporum f. sp. ciceris is one of the most economically important disease in the most chickpea-growing areas (Jimenez-Diaz et al., 2015), but yet there has been no formal report of the Fusarium wilt of chickpea in Serbia. In June 2018, the first symptoms of Fusarium wilt were registered at Rimski Šančevi (Vojvodina Province), Serbia (N 45019.311' E 019049.933') as wilted chickpea plants grouped in patches on approximately 5% plants in the area of 3 acres. Symptoms as yellowing and necrosis of foliage appeared as late wilt in the podding stage. Roots of affected plants showed no external discoloration, but cross-sections showed dark-brown discoloration of xylem tissue. To isolate the causal agent, symptomatic plants were collected, and ten cuttings of symptomatic root tissue were surface disinfected with 2% sodium hypochlorite solution for 5 min, rinsed three times in sterile distilled water, air-dried on sterilized filter paper and plated on potato dextrose agar (PDA) and water agar (WA) amended with streptomycin sulfate. After seven days of incubation at 25°C in the dark, isolates were preliminarily identified according to their morphological characters and Fusarium Laboratory Manual (Leslie and Summerell, 2006). For each isolate 100, micro/macroconidia and chlamydospores were measured. Conidia were hyaline; macroconidia sickle-shaped, with blunt ends, 2-4 septa (10.1-17.7 x 3.1-5.8 µm); microconidia ellipsoidal, 0-1 septa (4.9-8.6-2.7-3.5 µm). Chlamydospores were globose (4.3-8.8 µm). Representative isolates (K343, K375 and K378) were purified by a single-spore technique for further analyses (Leslie and Summerell, 2006). Molecular identification of three representative isolates (K343, K375 and K378) was made by sequencing the rRNA internal transcribed spacer (ITS) region and translation elongation factor 1α (TEF1) gen. For all isolates, the ITS and TEF1 genes were amplified and sequenced with primers ITS1/4 (White et al. 1990), EF1-728 and EF1-986 (Rehner and Buckley 2005). Based on a BLAST search of the NCBI nucleotide database, the ITS sequences (GenBank MK920204.1, MK928423.1 and MK928424.1) had 99.8% identity with F. oxysporum f. sp. ciceris isolate (MK074845.1). The TEF1 (GenBank MN788462.1, MN788463.1 and MN788464.1) had 96.3 -100% identities with F. oxysporum f. sp. ciceris isolate (FJ538245.1). The pathogenicity test was conducted on 7-day old plants according to the drench method described by Maitlo et al. (2016). The concentration of inoculation suspensions was adjusted on 1x106, and ten plants per isolate were tested and inoculated with 10 ml of suspension. Control plants were drenched with 10 ml of sterilized distilled water. Nine days after inoculation, first symptoms as leaves wilting and white mycelia presence around stem base occurred on plants inoculated with isolate K378. On day 11, the first symptoms occurred on plants inoculated with isolates K343 and K375 as well. Up to the 13th day after inoculation, all plants were wilted, and the pathogen was successfully reisolated and confirmed as F. oxysporum f.sp. ciceris. To the best of our knowledge, this is the first report of F. oxysporum f.sp. ciceris causing Fusarium wilt on chickpea in Serbia. <br> Acknowledgements <br>This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number: 451-03-9/2021-14/200032 <br>

First Report of Phyllosticta capitalensis Causing Black Freckle Disease on Rubus chingii in China.

Rubus chingii is used as an important traditional Chinese medicine, and belongs to the family Rosaceae. The fruit has multiple pharmacological activities, including antioxidant, anti-inflammatory, and improving cognitive impairment (Na Han et al. 2012). In June 2019, a new fungal infection was observed on the leaves of R. chingii in Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province, China, forming small lesions with reddish-brown edges along leaf veins. Over 500 plants were surveyed, and nearly 20% of the plants were symptomatic. The diseased plants grew poorly and appeared stunted, and severely affected plants died. Five symptomatic leaves were randomly collected from the field and washed with tap water and distilled water successively. The edges of infected leaf tissue were cut into small pieces (4 to 5 mm2), surface sterilized with 70% ethanol for 30 s and 0.1% HgCl2 for 1 minute, and then rinsed three times in sterile distilled water (Chen et al. 2016). The same fungus was isolated from 41 pieces. The hyphae of a representative isolate were gray, the colony surface was granular, the edges were uneven and white, and the culture turned black over time with black spherical conidia. Conidia were nearly elliptical, unicellular, and each with a hyaline, unstable apical appendage, 3 to 10 µm long. The size of conidia was 10 to 18 μm in length and 4 to 8 μm in width. These morphological characteristics are consistent with those described for the fungus Phyllosticta capitalensis. (Wikee et al. 2013). For an accurate identification, genomic DNA of a representative isolate of the pathogen was extracted to amplify the internal transcribed spacer (ITS) region, the transcription elongation factor (tefa-1), and actin (ACT) genes with the ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Cheng, L. L. ,et al. 2019), respectively. The ITS, tefa-1 and actin gene sequences were deposited in GenBank and assigned accession numbers MW308365, MW714380 and MW714381, respectively. BLAST search analysis of GenBank (NCBI) showed that the sequences had 100% similarity with those of Phyllosticta capitalensis (GenBank accession no. ITS, MN548091; tefa-1, MN958711; and ACT, MN565575). The pathogenicity of Phyllosticta capitalensis was verified using six healthy detached leaves from healthy R. chingii plants around 40 cm tall. A total of nine plants were used, and three leaves from each plant were artificially inoculated. Each wound was inoculated with conidial suspension (106 mL-1), while the control leaves were coated by sterile water. All the treated plants were covered with plastic bags for 2 days, incubated at 28ºC and 85% relative humidity, with a 12-hour photoperiod. After 15 days following inoculation, the injured leaves showed similar symptoms to the above-mentioned lesions, while the control and uninjured leaves were still healthy. P. capitalensis were reisolated from inoculated leaves, fulfilling Koch's postulates. P. capitalensis is an endophyte, widely distributed in various host plants in China. (Lu, J. M, et al. 2016). To the best of our known, this is the first report of black freckle disease caused by P. capitalensis on Rubus chingii in China. P. capitalensis is a destructive plant pathogen with an unusually broad host range and our findings will be useful for its management and for further research. The author(s) declare no conflict of interest.

Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia

Background and Aim:Bovine eimeriosis is a disease caused by apicomplexan parasites of the genus Eimeria. It is one of the most important and widespread bovine illnesses in the world. Some of the identified species of bovine eimeriosis have morphologically similar oocysts that are difficult to differentiate. For the identification of particular Eimeria spp., diagnostic laboratories are increasingly turning to DNA-based technology. This study aims to develop a multiplex polymerase chain reaction (mPCR) technique based on the internal transcribed spacer-1 (ITS-1) gene for the simultaneous identification of pathogenic Eimeria spp. in cattle from Sulawesi Island, Indonesia.Materials and Methods:Genomic DNA was extracted by the DNAzol reagent from the purified Eimeria oocysts. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp.Results:Using PCR ITS-1, this study showed that 36 of 120 fecal samples (30%) were infected by Eimeria spp. The multiplex PCR assay allowed for the simultaneous identification of six major Eimeria spp. in a single-tube reaction. The proportion of mixed Eimeria spp. infections was 100% (36/36). The maximum number of Eimeria spp. was five, and the minimum number was two.Conclusion:Identification of six pathogenic Eimeria spp. in cattle was successfully carried out by nested multiplex PCR using ITS-1 gene. In the future, a procedure to detect pathogenic Eimeria spp. in one tube reaction will offer economical and save diagnostic time.

Phylogenetic Analysis of the Dominant Non-biting Midge Forcipomyia bikanni (Diptera: Ceratopogonidae) around West Lake, Hangzhou, China

Abstract The nonbiting midges are tiny insects with a wide distribution around West Lake, China. In this study, the seasonal dynamics of the local dominant midge species was investigated. Phylogenetic trees of this species also were analyzed to discover its evolutionary history. Ultraviolet (UV) light traps were used for surveillance around West Lake in the period from April 2017 to November 2018. External morphological identification was performed using a stereomicroscope. Two genes of adult female midges that encoded the cytochrome oxidase subunit I (COI) and internal transcribed spacer 1 (ITS-1) were sequenced and aligned online using GenBank nucleotide database and the Molecular Evolutionary Genetics Analysis (MEGA) software 10.1.7. According to the field surveillance in 2017–2018, Forcipomyia bikanni Chan and LeRoux (Diptera: Ceratopogonidae) is the representative dominant species of West Lake, with its activity peaking in August and October. The COI and ITS-1 gene sequences of F. bikanni (XH01-5) were 523 bp and 315 bp, respectively. The final phylogenetic tree of the COI implied that F. bikanni (XH01-5) has 97.9% significant similarity with conspecific F. bikanni (LC015045) from the adjacent Fujian Province, China (E-value ≤ 0.1%). Furthermore, the ITS-1 sequences with 98% likelihood showed the highest linkage between F. bikanni (XH01-5) and F. townsvillensis (Taylor) (HM775502-HM775504) with different branches. Thus, the final unrooted phylogenetic trees of COI and ITS-1 are essential tools for reconstructing the evolutionary histories of nonbiting midges.

First Report of Fusarium proliferatum Causing Fruit Rot on Muskmelon (Cucumis melo) in China.

Muskmelon is an economically important crop in the world, especially in China, the largest producer of muskmelon with an annual output up to 12.7 million tonnes (Gómez-García et al. 2020). Since 2018, fruit rot was observed on muskmelon in Malianzhuang Base, the main muskmelon producing area in Shandong Province, whose disease incidence was about 25-30%. Water-soaked dark brown spots were initially appeared on the side of the fruit near the ground, then gradually expanded and covered with white mold with time. To isolate the pathogens, ten muskmelon fruits with typical symptoms were collected from different greenhouses in the base. Small tissues taken from the edge of the diseased and healthy tissues were immersed in 1% NaClO for 2 min, then soaked in 75% ethanol for 30 s, and rinsed 3 times with sterile distilled water (SDW). The sterilized tissues were naturally dried and placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/L) for 7 days at 28℃. The emerging fungal mycelia were transferred to fresh PDA using the hyphal tip technology. Ten colonies were purified by single spore method and cultured on PDA for 7 days at 28℃ in the dark for morphological and molecular analyses. All colonies were flocculent with abundant white to light purple aerial hyphae, and the undersides of the colonies were observed to be from white to purple over time. Microconidia produced on PDA were hyaline, fusiform, ovoid, single cell without septum, and 4.5 to 12.7 × 2.0 to 3.6 μm in size (n=50). Macroconidia produced on carboxymethylcellulose agar (CMC) were slightly curved at both ends with three to five septa, and 17.6 to 35.7 × 2.8 to 4.0 μm in size (n=30). According to the morphological characteristics, these isolates were preliminarily identified as Fusarium sp. (Leslie and Summerell 2006). To further identify these isolates, genomic DNA of five isolates was extracted by CTAB method (Wu et al. 2001). The internal transcribed spacer (ITS) region of ribosomal DNA, translation elongation factor 1-α (TEF1) region, and the RNA polymerase II second largest subunit (RPB2) were amplified by PCR amplification with primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F2/fRPB2-7cR, respectively (White et al. 1990; O'Donnell et al. 2008; Liu et al. 1999). Sequences of the five isolates were identical. The ITS, EF1-α, and RPB2 gene sequences of isolate NEAU-Mf-10-2 were submitted to NCBI GenBank with accession numbers of MZ950914, MZ960928, and MZ960929, respectively, having 100% similarity to those of Fusarium proliferatum (MK372368, MK952799 and MN245721). Phylogenetic trees were constructed based on the concatenated sequences of EF1-α and RPB2 genes using neighbour-joining and maximum-likelihood algorithms with MEGA 7.0. Two similar tree topologies both showed isolate NEAU-Mf-10-2 clustered with F. proliferatum NRRL 43665. Therefore, isolate NEAU-Mf-10-2 was identified as F. proliferatum based on morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, ten muskmelon fruits (var. Tianbao) were soaked in 2% NaClO for 2 min, and then washed three times with SDW. Muskmelon fruits were inoculated by injecting conidia suspension (200 μL, 1×106 spores/mL) with a sterile injector. Ten other surface sterilized muskmelon fruits inoculated with sterile water were used as control. The fruits were placed in a light incubator at 28℃ with 12h light cycles for 7 days. All inoculated fruits showed symptoms highly similar to those of infected muskmelon fruits observed in the field. No symptoms were observed on fruits used as control. The Fusarium isolates were successfully re-isolated from the symptomatic fruits, and identified based on above morphological and molecular biological methods. Previous studies have reported that F. proliferatum can infect Polygonatum cyrtonema, Salvia miltiorrhiza, Allium cepa, A. sativum, and so on. To our knowledge, this is the first report of F. proliferatum causing fruit rot on muskmelon in China, which will provide basic information for designing effective prevention and control strategies on this disease.

Differing Roles of Bacterial and Fungal Communities in Cotton Fields by Growth Stage

The global demand for cotton makes sustainable cotton production an important issue that can be improved by a better understanding of the influence of soil microbes on cotton growth. We collected cotton field soils at the seedling and flowering/boll-setting (FBS) stages in order to obtain soil properties and cotton growth indices. Bacterial and fungal community compositions were assessed by high-throughput sequencing of 16S rRNA and internal transcribed spacer genes, respectively, after which the differences in microbial functions and their influencing factors at different growth stages were analyzed. Both the diversity and composition of soil bacterial and fungal communities were found to be significantly different between the seedling and FBS stages. Microbes in the seedling stage had significantly higher richness and biomass than those in the FBS stage. Compared with the seedling stage, the stability of the soil bacterial communities was decreased. The cotton growth indices at both the seedling and FBS stages were associated with compositional shifts in the bacterial community and but not the fungal community. The abundance of specific soil microbial taxa (e.g., Pseudarthrobacter, Thiobacillus, Cephalotrichum, Chaetomium, and Fusarium) were correlated with cotton growth indices at the seedling stage, being mainly regulated by soil salinity and nitrate content. Our results highlight the importance of soil microbial communities in mediating cotton growth and will be useful in providing better strategies for the improvement of cotton agriculture.

Clarireedia hainanense: A New Species Is Associated with Dollar Spot of Turfgrass in Hainan, China.

The genus Clarireedia contains multiple species causing dollar spot (DS) on turfgrass worldwide. In November 2020, 119 Clarireedia isolates were obtained from symptomatic seashore paspalum at golf courses in Hainan province and identified to species level based on partial sequence of the internal transcribed spacer (ITS) region. A total of 45 and 22 isolates were identified as C. paspali and C. monteithiana, respectively; the remaining 52 isolates defined a new clade. Isolates from this clade were further selected for phylogenetic, morphological, and biological analyses. Maximum likelihood and Bayesian methods were implemented to obtain phylogenetic trees for partial sequences of the ITS, EF-1α, and McM7 genes. The selected isolates consistently fell into a distinct, well-supported clade within Clarireedia. Morphological and biological characteristics were observed among the different species in Clarireedia. Altogether, this study described a new species, Clarireedia hainanense, which has widespread distribution in Hainan, China. These findings may have important implications for the management of DS disease.

Haplotype diversity of palm weevil in Saudi Arabia through ITS gene sequencing

The red palm weevil, Rhynchophorus ferrugineus (Olivier 1790) caused a high economic loos in date palm in gulf region especially in Saudi Arabia. Previous estimations of palm weevil haplotype diversity in Saudi Arabia was not satisfactory because of choice of marker and small sampling area. Assessing haplotype diversity using COI gene was found ineffective. The present study was carried out to estimate the haplotypes of R. ferrugineus using internal transcribed spacer (ITS) sequences. Results showed 3 haplo-groups, among which, the haplotype UT9 was a singleton haplotype. The haplotypes UT16, UT2, UT6 UT10, UT14 and UT5 formed the second haplo-group whereas the remaining haplotypes were clustered as third group. K2P distance varied between 0 and 0.06 among the sequenced haplotypes and the maximum distance was recorded between two sets of haplotypes; viz; UT4 and UT3, UT4 and UT8. The overall distance of all the sequenced haplotypes were 0.02. Tajima’s neutrality value was < 0 indicating the potentiality for further haplotype diversification. Median joining haplotype network diagram reveals the presence of 10 haplotypes and most mutations were at 244th and 381st nucleotide positions. Overall, total number of mutations leading to the evolution of the documented 10 haplotypes was 34. Through genetic data, we have provided a strong evidence that there were multiple haplotypes of R. ferrugineus which were bound to expand its diversity. Higher nucleotide diversity and Tajima’s neutrality test values suggest that there were higher movement of infested palm trees, as desserts were natural barriers for movement of H. ferrugineus. The findings of this study will be useful for pest controlling agencies and to the workers involved in infested date palm quarantining.

Internal Transcribed Spacer (ITS) regions: A powerful tool for analysis of the diversity of wheat genotypes

Wheat is a widely cultivated crop and it is one of the major food sources worldwide. Among the various tools used to study diversity of wheat species, the internal transcribed spacer (ITS) assessment emerges to be the more appropriate approach. In the present study, we evaluated 15 genotypes of Iranian wheat cultivars (wild, native, and breed) using ITS gene sequences. Similarity matrices and dendrogram of phylogenic relationship were constructed using Mega ver6 software. We report the major nucleotide changes in the same position between diploid and hexaploid species. dN/dS ratio for diploid, tetraploid, and hexaploid species indicated a pure selection in the examined gene, with no key changes in the genes, and 91% ITS diversity within individual wheat was evident. The results suggest that as evolution moves forward, nucleotide changes are reduced so that only a few changes in nucleotides occur. ITS marker can distinguish different wheat genotypes at the genomic level and thus prove to be the most appropriate assessment tool for analyzing inter and intra-species relationships.

DNA barcoding for the discrimination of Uncaria gambir and its closely related species using internal transcribed spacer genes

Background: Uncaria gambir is one Uncaria species that exclusively grows in Indonesia. The phytochemical constituents of this species have been widely explored and its extracts are used as traditional medicine. However, the relationship between Uncaria gambir and other Uncaria species is still unknown. DNA barcoding was used in this study to reveal this relationship. Methods: Genomic DNA was isolated from four main cultivated variants of Uncaria gambir species in Indonesia. ITS primer was used to amplify the specific gene region. Genetic distance analysis was carried out on Uncaria gambir and 12 other Uncaria species. A phylogenetic tree was created to determine the relationship among Uncaria species using the maximum likelihood method. Results: The ITS primer successfully amplified the ITS region in Uncaria gambir. Genetic distance and phylogenetic tree analyses showed that Uncaria gambir has a close relationship with Uncaria scandens, Uncaria yunnanensis, and Uncaria macrophylla which is also indicated by Interspecific distance analysis. Conclusions: Although the DNA barcoding gap is absent in genetic distance analysis, the phylogenetic tree analysis from the ITS region can differentiate Uncaria gambir from other Uncaria species.

Antagonistic Activity of Fungal Strains against Fusarium Crown Rot.

The crown rot of wheat is a destructive soil-borne pathogen that severely reduces the yield and quality of wheat. This study aimed to screen and identify the antagonistic strains against Fusarium pseudograminearum (Fp), which is the dominant pathogen associated with the crown rot of wheat in China, and evaluate their biosynthetic potential. The antagonistic strains were screened via a dual-culture antagonism assay, and then identified by combining the morphological characteristics and internal transcribed spacer gene sequencing. The polyketide synthases (PKS-I and PKS-II) and non-ribosomal peptide synthetase (NRPS) genes in the antagonistic strains were detected via specific amplification of chromosomal DNA. Eleven out of 157 fungal strains, including six strains with matrix competition and five strains with antibiosis, were obtained. The eleven antagonistic strains belonged to the following four genera: Alternaria, Botryosphaeria, Phoma and Talaromyces. The inhibition rate of six strains with matrix competition was greater than 50%, with B. dothidea S2-22 demonstrating the highest at 80.3%. The width of the inhibition zone of T. trachyspermus R-17 among the five strains with antibiosis was the widest at 11 mm. Among the eleven antagonistic strains, three strains of A. alternata and the strain P. moricola only contained the PKS-II gene, the strain A. tenuissima contained PKS-I and PKS-II genes, three strains of B. dothidea contained PKS-II and NRPS genes, while three strains of T. trachyspermus did not contain any genes. These results demonstrated potential strains for the biocontrol of the crown rot of wheat. In particular, T. trachyspermus R-17 can be investigated further as a promising agent, and the active substances secreted by antagonistic strains may be synthesized by other pathways.

Association of Neofusicoccum parvum with leaf scorch on Cinnamomum cassia in China

AbstractLeaf spot, caused by Neofusicoccum parvum, is an important disease limiting the growth of Chinese cinnamon (Cinnamomum cassia) in the field. This disease was first observed in Deqing (Guangdong Province, China) in 2018, manifesting as circular or irregularly shaped necrotic lesions along the leaf margins and tips, enlarging to irregular lesions with numerous black dots. Representative isolates were isolated from affected leaves and identified based on morphological characteristics and phylogenetic analyses of the internal transcribed spacer (ITS) region, the translation elongation factor 1‐α (EF1‐α) gene and a portion of the β‐tubulin gene (TUB2). Colonies were initially white on PDA, turning grey–black after 7 days. The conidia were fusiform to oval, hyaline, thin‐walled with rounded apices and truncate bases. The pathogen was placed in the same clade as Neofusicoccum parvum in phylogenetic trees constructed using concatenated sequences of ITS, EF1‐α and TUB2 genes. Based on morphological and phylogenetic data, the pathogen was identified as N. parvum. On infected leaves, Koch's postulates were fulfilled, and the same pathogen re‐isolated. To our knowledge, this is the first report of N. parvum causing disease on Chinese cinnamon worldwide.

Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador.

Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2-gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.

Molecular and morphological characterization of stunt nematodes of wheat, maize, and rice in the savannahs of northern Nigeria

Stunt nematodes (Tylenchorhynchus spp.) are obligate migratory root ecto-parasitic nematodes found in the fields of many cultivated crops. These nematodes, with phyto-sanitary potential, are frequently ignored or misdiagnosed as pests, and this may pose a threat to food security. The accuracy of its identification based on a morphological approach has been challenged recently, due to the overlapping of the morphological and morphometric characters of the species. Consequently, the objective of this study is to identify and characterize stunt nematodes present in 54 fields cultivated with cereal crops (wheat, maize and rice) in the savannahs of northern Nigeria, using integrative taxonomy and molecular approaches. The molecular and morphological studies identified and confirmed the presence of T. annulatus as the occurring specie in the savannahs of northern Nigeria. The phylogenetic analysis was carried out using the internal transcribed spacer (ITS) and 28S genes of ribosomal DNA further confirmed the presence of T. annulatus. The first molecular characterization and sequences of the ITS and 28S rDNA gene for T. annulatus from Nigeria were provided by this research. Also, according to our literature search, this is the first report on T. annulatus in wheat, maize and rice in the savannahs of northern Nigeria. Further study to test the pathogenicity of the parasitic nematode species found in this survey is recommended for the prioritization and development of efficient management strategies.

Mitochondrial and nuclear DNA-based identification of some forensically important calliphoridae (diptera) in Luoyang of China

Introduction: Calliphoridae plays a key role in forensic entomology research, which is one of the first insects to decompose animal carcasses.The mitochondrial cytochrome c oxidase subunit I and the ribosomal internal transcribed spacer 2 (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Aim: The aim of the study was to test the suitability of two genetic markers based on conducting the molecular identification of six necrophagous Calliphorid flies. Materials and Methods: Fourteen Calliphoridae flies were collected and classified with traditional morphological characteristics. The DNA of flies was extracted and the fragments of COI and ITS2 were amplified and sequenced. All the sequences were aligned and analyzed by MEGA 7 software for NCBI BLAST, nucleotide composition, intra- and inter-specific divergence calculation, and phylogenetic tree inference successively. Results: The results indicated that COI and ITS2 genes were robust in the identification of Calliphoridae at the species level and ITS2 gene sequence possessed a strong resolution power as it showed higher variation values between Lucilia sericata and Lucilia cuprina, Calliphora vomitoria and Triceratopyga calliphoroides, C.vomitoria and Aldrichina grahami, but inferior to COI for T. calliphoroides and A. grahami. Conclusions: Our results showed that combination of COI + ITS2 genes yields more accurate identification and diagnoses and better agreement with morphological data than the mitochondrial barcodes alone. As a supplementary method for morphological identification, we advocated for the combination of nuclear and mitochondrial gene approaches to address the taxonomy and phylogeny of forensic relevant flies, especially of closely related species and populations.