Spacer Gene Research Articles

Phylogenetic Relationships of the Strongyloid Nematodes of Australasian Marsupials Based on Mitochondrial Protein Sequences

Australasian marsupials harbour a diverse group of gastrointestinal strongyloid nematodes. These nematodes are currently grouped into two subfamilies, namely the Cloacininae and Phascolostrongylinae. Based on morphological criteria, the Cloacininae and Phascolostrongylinae were defined as monophyletic and placed in the family Cloacinidae, but this has not been supported by molecular data and they are currently placed in the Chabertiidae. Although molecular data (internal transcribed spacers of the nuclear ribosomal RNA genes or mitochondrial protein-coding genes) have been used to verify morphological classifications within the Cloacininae and Phascolostrongylinae, the phylogenetic relationships between the subfamilies have not been rigorously tested. This study determined the phylogenetic relationships of the subfamilies Cloacininae and Phascolostrongylinae using amino acid sequences conceptually translated from the twelve concatenated mitochondrial protein-coding genes. The findings demonstrated that the Cloacininae and Phascolostrongylinae formed a well-supported monophyletic assemblage, consistent with their morphological classification as an independent family, Cloacinidae. Unexpectedly, however, the subfamily Phascolostrongylinae was split into two groups comprising the genera from macropodid hosts (kangaroos and wallabies) and those from vombatid hosts (wombats). Genera of the Cloacininae and Phascolostrongylinae occurring in macropodid hosts were more closely related compared to genera of the Phascolostrongylinae occurring in wombats that formed a sister relationship with the remaining genera from macropods. These findings provide molecular evidence supporting the monophyly of the family Cloacinidae and an alternative hypothesis for the origin of marsupial strongyloid nematodes in vombatid hosts that requires further exploration using molecular approaches and additional samples.

First Report of Neofusicoccum parvum Causing Leaf Spot on coral trees (Viburnum odoratissimum) in China.

Coral trees (Viburnum odoratissimum), as a class of evergreen shrubs, are mainly planted in landscapes in numerous cities in China. During September 2020, the author investigated four major parks in Hefei (Bao Park, Hefei Botanical Garden, Luzhou Park and Peninsula Park) and the campus of Anhui Agricultural University (approximately 0.5 ha) (31°49'21.30″N, 117°13'18.25″E). The results showed that the incidence rate of leaf spot disease reached 60% among approximately 100,000 coral trees planted in these areas. Coral trees begin to show leaf spots in August. In early stages of coral trees infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. After the disease patches expand and darken, the coral leaves eventually wither and fall, which seriously affects its viewing and admiring value. To identify the fungal pathogen, the five-point sampling method was used to take typical similar leaf samples from 5 regions, and 6 samples were taken from each site, so a total of 150 samples were obtained. Fragments of sample leaves were surface-sterilized with 1% NaClO, plated on potato dextrose agar, and incubated at 25 °C in the dark. A total of 275 strains were obtained from 150 samples. According to the morphological characteristics, 275 strains were purified and divided into four types. Four representative strains (MI1, K1, F1, D1) were selected from four types for further pathogenicity testing and identification. The pathogenicity test was conducted in triplicate by inoculating wounded leaves of 1-year-old potted V. odoratissimum with 20μL of a conidial suspension (106 conidia/mL). The control was inoculated with sterile water. The specimens were placed in a growth chamber while maintaining 90% relative humidity and 28℃. After five days, the characteristic lesions were observed only on inoculated MI1 spore suspension leaves. The same fungus was reisolated from the lesions, thus fulfilling Koch's postulates. The pathogenic fungi accounted for 60% of all strains. Fungal colonies were circular and had abundant white aerial mycelium, and colonies changed from white to pure black after maturity. Conidia were fusiform (16-17×5-6 μm), thin-walled, transparent, and without diaphragms. Molecular identification was performed by partially sequencing the internal transcribed spacer (ITS) gene, the translation elongation Factor 1-alpha(EF1-alpha) gene, and the β-tubulin (TUB2) gene by using the primers ITS1/ITS4 (White et al. 1990), EF1-728F (Alves et al. 2008)/EF1-986R (Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995), respectively. The obtained ITS sequence (MW767713) showed 99% identity with N. parvum CMW28429 (KU997429.1), the EF1-alpha sequence (MZ398261) showed 99% identity with N. parvum isolate A4 (FJ528597.1), and the TUB2 sequence (MZ398260) showed 99% identity with N. parvum isolate BO52 (KU554657.1). By combining the sequences of individual fragments of each fungus in the order ITS, EF1-alpha and TUB2, MEGA 6.0 was used to analyze the sequence of kinship by using the maximum likelihood method, and the repeat value of bootstraps was 1000. A polygenic phylogenetic tree analysis based on multilocus alignment (ITS, EF1-alpha and TUB2) was constructed with some strains of Botryosphaeriaceae species. The results of the phylogenetic tree showed that MI1 and N. parvum clustered into a branch. To our knowledge, this is the first report of N. parvum causing leaf spot on V. odoratissimum in China.

Characterization of microbial community assembly on new wooden vats for use in cheese production

Wooden vats are used in the production of some traditional cheeses as the biofilms on wooden vat surfaces are known to transfer large quantities of microbes to cheese. However, very few studies have investigated the microbial composition of biofilms on newly developed wooden vats and how communities assemble and evolve. In the present study, the microbial communities of biofilms were characterized over the activation process on new wooden vats using amplicon sequencing of bacterial 16s rRNA and fungal internal transcribed spacer genes. Results showed that microbes from the whey effectively developed on wooden vats. Lactococcus was highly dominant throughout the vat activation process with substantial increases in the relative abundance of Acetobacter and Lactobacillus at the end of the vat activation (day 7). This was in contrast with fungal communities that stabilized early (day 1) and were dominated by Kluyveromyces. Predicted functions corresponded with the different stages of biofilm formation whereby functions associated with biofilm initiation were enriched on day 1 and those associated with growth and maturation were enriched on days 4 and 7. Microbial succession on wooden vat surfaces is expected to be reproducible based on the early onset and dominance of the deterministic process.

Isolation and screening of entomopathogenic fungi against the grasshopper Oxya chinensis (Orthoptera: Acrididae) from rice fields in Thua Thien Hue, Vietnam

Abstract. Cuong LCV, Souvannalath B, Ha TP, Lien LQ, Tam VTT, Hoai PTT, Anh HLT, Dat TTH. 2022. Isolation and screening of entomopathogenic fungi against the grasshopper Oxya chinensis (Orthoptera: Acrididae) from rice fields in Thua Thien Hue, Vietnam. Biodiversitas 23: 4906-4911. The grasshopper Oxya chinensis is one of the most abundant herbivorous insects of rice plants in Vietnam and causes severe damage to the crop. Therefore, biological control has become a particularly valuable method for grasshopper and insect control in agricultural production. The aim of the present study was to isolate and investigate the potential entomopathogenic fungi against the grasshopper O. chinensis from rice fields in Thua Thien Hue, to develop environmentally friendly bio-insecticides. The results showed that 37 fungal isolates were isolated from dead grasshoppers collected from rice fields in Thua Thien Hue province. Results of pathogenicity screening of 12 selected fungal isolates showed that the mortality rate of 4th-instar grasshopper nymphs O. chinensis ranged from 21.99% to 100% after 15 treatment days at a concentration of 1 × 108 conidia/ml. Of these, three fungal isolates, namely NL3, NL4, NL12 showed the highest pathogenicity against the grasshopper O. chinensis with mortality rates of 87.73%, 89.81%, and 100%, respectively after 15 days. LC50 values of NL3, NL4, NL12 isolates against 4th-instar nymphs and adults of O. chinensis ranged from 9.5×104 to 4.8×105 conidia/mL and 7.8 × 106 to 2.5 × 106 conidia/mL, respectively at 7 days. LT50 values of NL3, NL4, NL12 isolates against 4th-instar nymphs and adults of O. chinensis were from 4.1 to 4.9 days and 5.5 to 6.1 days, respectively at a concentration of 1 × 108 conidia/mL. Results from morphological identification and nuclear ribosomal internal transcribed spacer (ITS) gene sequence showed that three potential entomopathogenic fungi were Aspergillus tamarii, Beauveria bassiana, and Metarhizium anisopliae. The pathogenicity of Aspergillus tamarii, Beauveria bassiana, and Metarhizium anisopliae against the grasshopper O. chinensis is reported for the first time. The present investigation revealed that these entomopathogenic fungi could be a useful source for developing environment-friendly bio-insecticides for sustainable agricultural production.

P276 Molecular epidemiology of clinical filamentous fungi in Qatar beyond Aspergillus and Fusarium

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectivesDue to an increasing number of patients at risk, (ie, with a highly compromised immune system and/or receiving aggressive chemotherapy treatment), invasive fungal infections (IFI) are increasingly being reported. They are associated with significantly high mortality rates. Aspergillus spp., particularly A. fumigatus, is the major cause of mold-related IFI around the world followed by Fusarium spp., however, other molds are emerging as human pathogens. The aim of this study was to explore the epidemiology and prevalence of the non-Aspergillus and non-Fusarium molds in human clinical samples over 11 years period in Qatar based tertiary hospital using molecular techniques.MethodsA total of 91 clinical specimens positive for molds belonging to 90 patients were recorded in about 11 years (September 2003 to November 2014). The isolates were identified based on morphological characteristics and by sequencing the internal transcribed spacer (ITS) gene. To confirm the identifications, a phylogenetic tree based on ITS sequences was constructed.ResultsMost patients were males (72%), 6% were immunocompromised, 12% had IFI, and 7% died within 30 days of diagnosis. The fungal isolates were recovered from a variety of clinical samples, including nails, skin, hair, scalp, nasal cavity, wounds, respiratory samples, body fluids, eye, ear, tissue, abscess, and blood specimens. Dematiaceous fungi were overall the most isolated (33/91, 36%), followed by dermatophytes (25/91, 27%), Mucorales (16/91, 18%), and other hyaline molds (17/91, 19%) (Fig. 1). Curvularia was the most isolated genus (22/91, 24%) and Mucorales were the major cause of IF (5/11, 45.5%) (Table 1). Superficial mycoses were caused by dermatophytes (66%) and non-dermatophytes (34%). Furthermore, rare fungi that are not commonly known to cause human disease were recovered such as Rhytidhysteron rufulum, Subramaniula asteroids, Dothichiza pimprina, and Quambalaria cyanescens.ConclusionThe current study highlights the epidemiology and spectrum of mold genera, other than Aspergillus and Fusarium, recovered from human clinical samples in Qatar, which can aid in surveillance of uncommon and emerging mycoses other than aspergillosis and fusuriosis.

Prevalence and Molecular Identification of Schistosoma haematobium among Children in Lusaka and Siavonga Districts, Zambia.

Schistosomiasis remains a public health concern in Zambia. Urinary schistosomiasis caused by Schistosoma haematobium is the most widely distributed infection. The aim of the current study was to determine the prevalence and risk factors of urinary schistosomiasis and identify the strain of S. haematobium among children in the Siavonga and Lusaka districts in Zambia. Urine samples were collected from 421 primary school children and S. haematobium eggs were examined under light microscopy. A semi-structured questionnaire was used to obtain information on the socio-demographic characteristics and the potential risk factors for urinary schistosomiasis. DNA of the parasite eggs was extracted from urine samples and the internal transcribed spacer gene was amplified, sequenced and phylogenetically analysed. The overall prevalence of S. haematobium was 9.7% (41/421) (95% CI: 7.16–13.08), male participants made up 6.2% (26/232) (95% CI: 4.15–9.03), having a higher burden of disease than female participants who made up 3.5% (15/421) (95% CI: 2.01–5.94). The age group of 11–15 years had the highest overall prevalence of 8.3% (35/421) (5.94–11.48). Participants that did not go fishing were 0.008 times less likely to be positive for schistosomiasis while participants whose urine was blood-tinged or cloudy on physical examination and those that lived close to water bodies were 9.98 and 11.66 times more likely to test positive for schistosomiasis, respectively. A phylogenetic tree analysis indicated that S. haematobium isolates were closely related to pure S. haematobium from Zimbabwe and hybrids of S. haematobium × S. bovis from Benin, Senegal and Malawi. The current study shows that urinary schistosomiasis is endemic in the study areas and is associated with water contact, and S. haematobium isolated is closely related to hybrids of S. bovis × S. haematobium strain, indicating the zoonotic potential of this parasite.

MOLECULAR IDENTIFICATION AND GERMPLASM CONSERVATION OF WILD MUSHROOMS FROM MAYANTOC, TARLAC, PHILIPPINES

Tarlac is a landlocked province located in Central Luzon, Philippines. It has natural forests containing natural vegetation which offers favorable surroundings for the mushrooms to grow. In this study, a collection of macrofungi was conducted, followed by molecular identification. The genomic DNA was extracted through CTAB method, and amplified the ITS region using Internal Transcribe Spacer (ITS) gene marker. The amplified products were sequenced and identified using BLAST NCBI analysis. There are 25 mushroom samples that were successfully identified and with high similarity with those of other related species retrieved from GenBank. The samples were classified into 6 orders, 12 families, 15 genera, and 25 species from Basidiomycota and Ascomycota which separated into two branches in the phylogeny tree. The tree also shows that the most species collected were from the order Polyporales with 11 species grouped in the first clade followed by Agaricales with 8 mushroom species. A portion of the fruiting body with 15% glycerol and all the mycelial samples were transferred in cryovials and placed in a cryofreezer for preservation.

Enzyme-Mediated Amplification (EMA) for Detection of the Pinewood Nematode Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a notorious parasitic nematode of pine trees that causes pine wilt disease (PWD), leading to extensive mortality of different pine species around the world and considerable economic losses, thus posing a threat to healthy pines worldwide. Fast and accurate detection technology is necessary for the management of PWD spread. This study describes the development of a new DNA extraction method and detection technology, enzyme-mediated amplification (EMA), using primers and a newly designed probe according to the rRNA internal transcribed spacer gene ITS2. The detection process can be completed within 40 min, including DNA extraction for 10 min and detection for 30 min, by exploiting the synergistic action of multiple enzymes. This method can detect PWNs from different geographic areas quickly and accurately at all life stages, singly or in a mixture, and can distinguish PWNs from other species of the Bursaphelenchus group, showing that it is not only reliable but also rapid, greatly improving the efficiency and speed of PWN detection. Therefore, the technology is expected to be highly beneficial in PWN quarantine testing.

Molecular prevalence and genetic diversity of Bartonella spp. in stray cats of İzmir, Turkey

BackgroundBartonella spp. are vector-borne pathogens that cause zoonotic infections in humans. One of the most well-known of these is cat-scratch disease caused by Bartonella henselae and Bartonella clarridgeiae, with cats being the major reservoir for these two bacteria. Izmir, Turkey is home to many stray cats, but their potential role as a reservoir for the transmission of Bartonella to humans has not been investigated yet. Therefore, the aim of this study was to investigate the prevalence of Bartonella species and their genetic diversity in stray cats living in Izmir.MethodsMolecular prevalence of Bartonella spp. in stray cats (n = 1012) was investigated using a PCR method targeting the 16S-23S internal transcribed spacer gene (ITS), species identification was performed by sequencing and genetic diversity was evaluated by haplotype analysis.ResultsAnalysis of the DNA extracted from 1012 blood samples collected from stray cats revealed that 122 samples were Bartonella-positive, which is a molecular prevalence of 12.05% (122/1012; 95% confidence interval [CI] 10.1–14.2%). Among the Bartonella-positive specimens, 100 (100/122; 81.96%) were successfully sequenced, and B. henselae (45/100; 45%), B. clarridgeiae (29/100; 29%) and Bartonella koehlerae (26/100; 26%) were identified by BLAST and phylogenetic analyses. High genetic diversity was detected in B. clarridgeiae with 19 haplotypes, followed by B. henselae (14 haplotypes) and B. koehlerae (8 haplotypes).ConclusionsThis comprehensive study analyzing a large number of samples collected from stray cats showed that Bartonella species are an important source of infection to humans living in Izmir. In addition, high genetic diversity was detected within each Bartonella species.

Clonostachys spp., natural mosquito antagonists, and their prospects for biological control of Aedes aegypti.

Aedes aegypti (Linnaeus, 1762) is an important vector of arboviruses in the tropics and subtropics. New control strategies based on natural enemies such as entomopathogenic fungi are of utmost importance, and the present study reports the first isolation of Clonostachys spp. (Hypocreales: Bionectriaceae) from mosquitoes and their activity against A. aegypti. Entomopathogenic fungi were surveyed in central Brazil using A. aegypti larvae as sentinels and, also, a CDC light trap. Clonostachys eriocamporesii R.H. Perera & K.D. Hyde, 2020 (IP 440) and Clonostachys byssicola Schroers, 2001 (IP 461) were identified by sequence analysis of the nuclear ribosomal internal transcribed spacer gene, and tested against eggs, larvae, and adults. Both strains were highly active against A. aegypti third instar larvae, with mortalities ≥ 80% at 107 conidia/mL after 5days but distinctly less active against eggs and adults. This is the first report of both C. eriocamporesii and C. byssicola as naturally occurring pathogens affecting mosquitoes, and IP 440 appears to be a promising control agent against aquatic stages of A. aegypti.

First Report of Colletotrichum siamense causing anthracnose on Podocarpus macrophyllus in Sichuan Province of China.

Podocarpus macrophyllus (Thunb.) D. Don is a perennial evergreen tree of the Podocarpaceae family, which is widely used in landscape, medicine and forest interplanting (Qin et al. 2021). In August 2020, approximately 10% of the leaves have expressed symptoms of anthracnose in the campus of Sichuan Agricultural University (E103°51'35.88″, N30°42'30.41″). The lesions were light brown small sunken spots on the leaf tip in the early stage, then spread along the petiole to expanded into larger, irregular gray-white lesions in the late stage, with sparse black dots arranged above. The edge of the lesion was obvious with a fine smooth dark brown line. Samples taken from the lesions were surface disinfected for 3 min in 4% sodium hypochlorite, rinsed in sterile water and plated on potato dextrose agar (PDA), Eight single-spore cultures isolates from 10 samples were obtained and subcultured. After five days at 25°C in the dark, the mycelium of a representative culture LJS1 covered the entire plate surface (9 cm diameter). Hyphae were initially white at first, and turned pale grayish in the later stage. After about 10 days, a large number of pink conidial mass were formed around the center. Conidia 14.7 - 18.6 μm (mean 16.2 μm) in length and 4.4 -7.1 μm (mean 5.8 μm) in width (n = 100), nonseptate, cylindrical, two ends round or one end slightly acute. Conidial appressoria 5.7 - 9.3 μm (mean 7.8 μm) in length and 4.4 - 7.9 μm (mean 6.2 μm) in width (n = 50), clavate, ovoid to slightly irregular. Based on these characteristics, isolates were tentatively identified as Colletotrichum siamense complex (Sharma et al. 2013). Pathogenicity tests were conducted by spraying a conidial suspension of LJS1 (1 × 107conidia/ml) to 10 wounded and 10 non-wounded leaves from P. macrophyllus plants. Two areas of cuticle on either side of the midrib of each leaf were wounded by lightly scratching with a needle prior to inoculation (Du et al. 2020). As a control, distilled water was sprayed onto an equal number of wounded and non-wounded leaves. All inoculated and control plants were incubated in greenhouse (about 25 ± 2°C). Lesions similar to those observed in the field appeared on all wounded inoculated leaves within eight days after inoculation, whereas the non-wounded inoculated leaves and the controls remained symptomless. Reisolations of the putative pathogen were confirmed through morphological characteristics and the representative culture LJS1 were confirmed to be the pathogenic agents. The internal transcribed spaces (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), gene spacer region between Apn2 and Mat1-2(ApMat) genes were sequenced (Sharma et al. 2013) and deposited in GenBank (accession numbers OK036793, OK067325 and OK086086 respectively). These sequences were highly identical to those of C. siamense Prihastuti, L. Cai & K.D. Hyde (culture LF 139): accession numbers KJ955087.1 (99%), KJ954788.1 (99%), KJ954503.1 (99%), respectively. Based on the morphology and our multi-locus approach, the pathogen was convincingly identified for the first time as C. siamense. However, there are no reports of C. siamense causing anthracnose on P. macrophyllus worldwide. The identification of the causal agent of the disease made clear the pathogen causing anthracnose on P. macrophyllus, and provide theoretical basis for the diagnosis and treatment of disease.

Growth Stimulation, Phosphate Resolution, and Resistance to Fungal Pathogens of Some Endogenous Fungal Strains in the Rhizospheres of Medicinal Plants in Vietnam.

Endophytic fungi are recognized for their many potential applications in agriculture, such as supporting cropland expansion and increasing the yield and resistance of plants by creating antibiotics that inhibit the growth of pathogenic microorganisms. In addition, they can produce enzymes that break down hard-to-solubilize substances within soil, dissolve phosphates, fix nitrogen, reduce metals, and produce hormones that promote plant growth (auxin, cytokinin, and gibberellins) to keep crops healthy. In this report, three strains of endophytic fungi, namely, N1, N2, and N3, were isolated from the roots of Stevia rebaudiana (Bert.) Hemsl., Polyscias fruticosa, and Angelica dahurica in some localities in Vietnam. Through a screening process, it was found that they can produce high levels of indole acetic acid (IAA), resolve phosphates, and resist disease, and they were selected to as an alternative to chemical fertilizers to make probiotics in order to increase medicinal plant yields. The results show that the three strains of fungi have the ability to degrade phosphate to 341.90, 1498.46, and 390.79 ppm; the content of IAA produced in the culture medium reached 49.00, 52.35, and 33.34 ppm. Based on some morphological characteristics and an internal transcribed spacer gene sequence analysis of the fungal strains, N1, N2, and N3 were named Penicillium simplicissimum CN7, Talaromyces flavus BC1, and Trichoderma konilangbra DL3, respectively, which have the ability to inhibit the growth of pathogenic fungal strains, such as fungus C. gloeosporioides (CD1), fungus F. oxysporum, fungus L. theobromae N13, and N. dimidiatum. They grow significantly over a period of 5 to 6 days.

First Report of Maize Seedling Blight Caused by Fusarium pseudograminearum in China.

Maize (Zea mays L.) is one of the most important food and feed crops in China, with a cultivation area of more than 40 million hectares (http://www.fao.org/faostat/en/#data/QC). In July 2021, a serious maize seeding blight occurred in Changjia Town, Gaoqing Country, Zibo City, Shandong Province, China, and the disease incidence was up to 50% in some fields. The root system of infected plants displayed poor development. The primary roots were brown and rotted. The leaves at the base of the plants were drying up, then the whole plant withered. To determine the cause agent of the disease, symptomatic roots of diseased seedlings were collected and surface-sterilized (70% ethanol for 30 s and 3% sodium hypochlorite (NaClO) for 90 s), subsequently rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), then incubated at 25°C for 2 days. Two cultures with similar morphological characteristics were purified through single-spore isolation technique and identified by morphology and molecular methods as Fusarium pseudograminearum O'Donnell & T. Aoki 1999. Plentiful macroconidia formed in 5-day-old carboxymethyl cellulose (CMC) cultures; microconidia were absent. Macroconidia were thick-walled and curved, usually 3- to 5- septa, 31.6 ± 0.6 μm × 4.8 ± 0.1 μm (n = 50). Colony pigmentation on PDA was pink to red, with white to pink aerial mycelia on PDA cultures was abundant and filled the petri dishes. For molecular identification, the rDNA internal transcribed spacer (ITS) gene and translation elongation factor 1 alpha (TEF-1α) gene of two isolates (SAIA41B and SAIA41C) were amplified with ITS1/ITS4 (White et al., 1990) and EF-1/EF-2 (O'Donnell et al., 1998), respectively. Blastn analysis of both the ITS sequence (accession numbers OM108101 and OM108102) and TEF-1α sequence (accession numbers OM142205 and OM142206) revealed 100% (481/481 bp for ITS and 637/637 bp for TEF-1α) sequence identity with the sequences of F. pseudograminearum reported in GenBank (MW699613 for ITS and JN862232 for TEF-1α). The molecular identification was further confirmed by the F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell 1999). The expected 523-bp fragments were obtained for isolates SAIA41B and SAIA41C. In the pathogenicity test, healthy germinating maize roots (Zhengdan958) were inoculated with PDA culture blocks of isolate SAIA41C. Plants inoculated only with PDA culture blocks served as controls. Maize plants were put in petri dishes and placed in an incubator with a 12-h photoperiod at 25 oC and 100% relative humidity. Seven days later, roots of the plants inoculated with isolate SAIA41C were poorly developed and became brown necrotic and rotted, which were identical to the symptoms observed in the fields, whereas the roots of control plants were developed normally. The pathogen was re-isolated from the necrotic tissue of the inoculated roots but not from the control plants, and its identity was confirmed by PCR with the primes Fp1-1/Fp1-2 described above, fulfilling Koch's Postulates. To our knowledge, this is the first report of maize seedling blight caused by F. pseudograminearum in China. Our finding indicates the potential spread of F. pseudograminearum on maize, and more attention should be paid to prevention and control of maize seedling blight caused by F. pseudograminearum. The author(s) declare no conflict of interest. Acknowledgements: This research was supported by National Natural Science Foundation of China (No. 32102181), Shandong Provincial Natural Science Foundation (No. ZR2021QC059), Wheat Industry Technology System of Shandong Province (No. SDAIT-01-10), and Agricultural Science and Technology Innovation Project of SAAS (No. CXGC2021A38 and CXGC2021A33).

Diversity of endophytic fungi in Coptis chinensis Franch. and their activity against methicillin-resistant Staphylococcus aureus

This study aimed at investigating the diversity of endophytic fungi from Coptis chinensis and their activity against methicillin-resistant Staphylococcus aureus (MRSA). Seventy-nine fungal isolates obtained from C. chinensis were identified to belong to 27 species based on morphological features and internal transcript spacer (ITS) gene sequencing analysis. Comparing relative frequency values, the most frequent genera were Colletotrichum and Fusarium, while most frequent species were C. gloeosporioides and F. avenaceum. Analysis of diversity indices indicated that C. chinensis harbored abundant fungal resources. Methanol extracts of fungal endophyte cultures were evaluated for antibacterial activity against S. aureus ATCC 25923 and two other MRSA clinical strains. Nine of 27 endophytic fungi exhibited inhibitory activities against S. aureus ATCC 25923. Among them, Paraboeremia litseae HL-17, Fusarium sp. HL-23, and Fusarium sp. HL-27 exhibited obvious inhibition against the three S. aureus strains. Our findings suggest that the endophytic fungi in C. chinensis have a high diversity and an obvious tissue specificity, and could be of potential interest in screening anti-MRSA agents. To the best of our knowledge, this is the first report on the diversity and anti-MRSA activity of fungal endophytes from C. chinensis.

Biogas slurry application alters soil properties, reshapes the soil microbial community, and alleviates root rot of Panax notoginseng.

BackgroundPanax notoginseng is an important herbal medicine in China, where this crop is cultivated by replanting of seedlings. Root rot disease threatens the sustainability of P. notoginseng cultivation. Water flooding (WF) is widely used to control numerous soilborne diseases, and biogas slurry shows positive effects on the soil physiochemical properties and microbial community structure and has the potential to suppress soilborne pathogens. Hence, biogas slurry flooding (BSF) may be an effective approach for alleviating root rot disease of P. notoginseng; however, the underlying mechanism needs to be elucidated.MethodsIn this study, we conducted a microcosm experiment to determine if BSF can reduce the abundance of pathogens in soil and, alleviate root rot of P. notoginseng. Microcosms, containing soil collected from a patch of P. notoginseng showing symptoms of root rot disease, were subjected to WF or BSF at two concentrations for two durations (15 and 30 days), after which the changes in their physicochemical properties were investigated. Culturable microorganisms and the root rot ratio were also estimated. We next compared changes in the microbial community structure of soils under BSF with changes in WF and untreated soils through high-throughput sequencing of bacterial 16S rRNA (16S) and fungal internal transcribed spacer (ITS) genes amplicon.ResultsWF treatment did not obviously change the soil microbiota. In contrast, BSF treatment significantly altered the physicochemical properties and reshaped the bacterial and fungal communities, reduced the relative abundance of potential fungal pathogens (Fusarium, Cylindrocarpon, Alternaria, and Phoma), and suppressed culturable fungi and Fusarium. The changes in the microbial community structure corresponded to decreased root rot ratios. The mechanisms of fungal pathogen suppression by BSF involved several factors, including inducing anaerobic/conductive conditions, altering the soil physicochemical properties, enriching the anaerobic and culturable bacteria, and increasing the phylogenetic relatedness of the bacterial community.ConclusionsBSF application can reshape the soil microbial community, reduce the abundance of potential pathogens, and alleviate root rot in P. notoginseng. Thus, it is a promising practice for controlling root rot disease in P. notoginseng.

No evidence for host plant associated genetic divergence in a population of Bullacris unicolor (Linnaeus, 1758) (Orthoptera: Pneumoridae)

Host-associated genetic differentiation in grasshoppers has received limited attention, due to a lack of information on grasshopper-plant associations. The bladder grasshopper, Bullacris unicolor (Linnaeus, 1758) (Orthoptera: Pneumoridae), is a phytophagous species that can occur on at least six host plants within its geographic range. However, the relationship between host plant association and genetic variation of bladder grasshoppers has not been studied before. In light of this, the present study examined host plant-related genetic [mitochondrial cytochrome oxidase 1 (CO1) and the internal transcribed spacer (ITS) gene regions] and morphological (antennal length, body length, head width, abdomen width, femur length, tibia length and pronotum length) divergence within a population of B. unicolor. We used two plant species, belonging to different families, namely Didelta spinosa (L.fil.) Aiton (Asteraceae) and Roepera morgsana (L.) Beier & Thulin (Zygophyllaceae), to evaluate variation between individuals collected on these two sympatric host plants at a single locality in the Northern Cape, South Africa. The results demonstrated non-significant host related genetic variation with very low values of FST, indicating a low level of variation. The phylogram strongly indicated that there are no host-associated genetic differences in B. unicolor by displaying limited genomic clustering, whereas some differentiation was observed between the morphological measurements of males and females among host plants. Further studies using microsatellite molecular markers may help to discern population genetic structure. In addition, significant host-associated morphological divergence highlights the need to examine the mechanisms by which host utilization affects morphological features.

Development of PCR-Based Assays for Rapid and Reliable Detection and Identification of Canker-Causing Pathogens from Symptomatic Almond Trees.

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, β-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures.

First results on the genetic variation of the Bulgarian populations of Morimus asper funereus (Mulsant, 1862) (Coleoptera, Cerambycidae)

There is an ambiguity about the taxonomic status of Morimus asper funereus (Mulsant, 1862), a saproxylic longhorn beetle, widespread in Bulgaria and other Balkan countries. It is a protected species under the Habitats Directive (Annex II) and under the Bulgarian Biodiversity Act. A recent molecular study, based on the cytochrome C oxidase subunit I (COI) and the second internal transcribed spacer (ITS2) gene se­quences, found that all European and Turkish populations of Morimus should be referred to M. asper (Sulzer, 1776) (Solano et al. 2013). The territory of Bulgaria and some other Balkan countries remain as an unstudied gap in that research. Here we present preliminary results on the genetic divergence and diversity of Morimus populations on the Balkans based on COI gene sequences using material from Bulgaria and Albania and the haplotypes’ sequences of Solano et al. (2013) obtained from GenBank. The material collected from Bulgaria аnd two localities in Albania was identified based on morphology characteristics as M. asper funereus (44 samples), M. verecundus bulgaricus Danilevski et all., 2016 (1 sample from the type locality), Morimus orientalis Reitter, 1894 (1 sample from Strandzha Mts.) and Lamia textor (Linnaeus, 1758) (1 sample) used as an outgroup. All obtained sequences were analyzed and haplotype diversity was estimated. The results show that Bulgarian populations of Morimus demonstrate relatively high haplotype diversity in correspondence to the diversity of the other European populations. Also, a phylogeographical hypothesis of the linkage between Bulgarian and other Balkan populations was developed. Four main lineages of divergence were identified. In addition, our results support the assumption that M. verucundus is a tentative subspecies of the morphologically and genetically variable M. asper.

Development of a Taqman Real-Time PCR for the Identification of Haemaphysalis longicornis (Acari: Ixodidae).

Haemaphysalis longicornis Neumann, a vector of various pathogens with medical and veterinary importance, is a recent invasive species in the United States. Like many tick species, discerning H. longicornis from congeners can be a challenge. To overcome the difficulty of morphological identification, a Taqman quantitative real-time PCR based on the internal transcribed spacer gene (ITS2) was developed for quick and accurate identification of H. longicornis with a detection limit of as low as 19.8 copies. We also applied the assay to 76,004 archived ticks and found 37 ticks were H. longicornis. One H. longicornis was submitted from Warren, Somerset County, New Jersey in June 2015, 2 yr earlier than the initial report from the United States. None of these 37 H. longicornis was positive for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, B. miyamotoi, B. mayonii, Babesia microti, or Ehrlichia muris–like agent.

Assessment of the antioxidative response and culturable micro-organisms of Lygeum spartum Loefl. ex L. for prospective phytoremediation applications

Abundant plant species in arid industrial areas are mining phyto-resources for sustainable phyto-management. However, the association with their rhizosphere is still poorly known for phytoremediation purposes. This study aims to assess the heavy metals (HMs) and metalloids uptake of Lygeum spartum Loefl. ex L. growing in cement plant vicinity and screen associated culturome for potential phytoremediation use. Bioaccumulation factor (BAF), the translocation factor (TF), and the mobility ratio (MR) were studied along with four sites. Lipid peroxidation (MDA), free proline (Pro), Non-protein thiols (NPTs), and reduced glutathione (GSH) were tested for evaluating the plant antioxidative response. Bacteria and fungi associated with L. spartum Loefl. ex L. were identified by 16S rRNA and fungal internal transcribed spacer (ITS1-ITS2) gene sequencing. Our results showed an efficient uptake of As, Pb, and Zn and enhanced GSH (0.34 ± 0.03) and NPTs (528.7 ± 14.4 nmol g−1 FW) concentrations in the highly polluted site. No significant variation of Arbuscular Mycorrhizal Fungi (AMF) was found. Among 29 bacterial isolates, potential bioremediation were Bacillus simplex and Bacillus atrophaeus. Thus, L. spartum Loefl. ex L. and its associated microbiota have the potential for phytoremediation applications. Novelty statement: This work has been set in line with the investigation of the integrative biology of Lygeum spartum Loefl ex L. and the screening of its associated microbiome for potential phytoremediation applications. This work is the first work conducted in a cement plant vicinity investigating the associated fungi and bacteria of L. spartum Loefl. ex L. and been part of a sectorial research project since 2011, for assessing the impact of industrial pollution and recognizing the accumulation potential of plant species for further phyto-management applications.