Rotted Tissue Research Articles

Whole genome sequencing of Leuconostoc suionicum and L. pseudomesenteroides isolates extracted from sugar beet roots.

Leuconostoc suionicum and Leuconostoc pseudomesenteroides are important lactic acid bacteria identified in rotted tissues of roots in the field and stored sugar beets. Here, we announce the genomes of L. suionicum and L. pseudomesenteroides, isolated from post-harvest sugar beet roots from Idaho and Minnesota.

Characterization and identification of Burkholderia gladioli causing stalk rot of sweet corn in Thailand

AbstractStalk rot disease is becoming an important disease in sweet corn crops because it causes significant yield losses of the crop. In July 2022, typical symptoms of stem rot were observed in fields of Mae Hia Agricultural Research, Demonstration and Training Center in Chiang Mai province of Thailand. The symptoms exhibited by affected crop included circular water‐soaked lesions, rot and necrotic tissue at the base of the stalk. Koch's postulates were fulfilled with reisolation of the inoculated strain from the inoculated tissues, and confirmation was obtained by phenotypic characters and 16S rRNA sequences. The isolated colony on nutrient agar was cream‐coloured, circular, with a convex elevation, entire margin and smooth surface; the optical character was translucent and exuded a yellowish pigment that diffused in the nutrient agar, gram‐negative, lacked arginine dihydrolase, occurrence of the oxidase and catalase reaction. The strains were able to utilize d‐galactose and glucose but were unable to utilize d‐maltose, d‐cellobiose, d‐fructose, d‐mannose, mannitol, lactose and sucrose. The strains showed positive results for gelatine liquefaction test, citrate utilization and casein hydrolysis, but starch hydrolysis and indole production were negative result. These characteristics are the same as those described previously for Burkholderia gladioli. Disease severity in sweet corn was 43.8%, with infections showing spots with dark margins and soft rot on mature leaves and flag leaves. Disease severity in waxy corn and maize were 21.9% and 15.6%, respectively, showing a few light or dark brown spots at the infection sites. Therefore, this represents the first report of leaf stripe and stalk rot disease in sweet corn in Thailand.

Morphological, molecular biology and pathological characterization of fungi causes root rot on grapevine

Background Grapevine is subjected to attack by several of soilborne fungi causing root-rot diseases that lead to limit the production of grapevine. Objective This study aimed to investigate the morphological, molecular identification, and pathogenic potential of fungi that causes root-rot disease of grapevine in Egypt. Materials and methods Isolation of fungi causing root rot of grapevine plants from the samples of diseased roots of different cultivars, i.e., crimson, superior, and flame seedless. Identification according to characterizations of morphological, cultural, and molecular biology based on internal-transcribed spacer 1 (ITS1). Pathogenicity tests of fungal isolates on grapevine plants under greenhouse conditions. Root-rot incidence, disease severity, and plant growth characteristics were determined. Results and conclusion Isolation traits from affected grapevine trees with different grape varieties in Egypt’s El-Nobaria Province, El-Behira Governorate, yielded eighteen isolates of four fungal genera. Fusarium spp. was the most fungal genus highly frequent on all cultivars tested and colonization (100%) on root-rot tissue of superior and flame-seedless cultivars, then (75%) on Crimson cultivar. While Lasiodiplodia spp. was recorded with moderate frequency and high colonization (100%) on rotten tissue of Crimson cultivar followed by (75%) in both flame-seedless and superior cultivars. As opposed to that, fungi of Macrophomina phaseolina followed by Rhizoctonia solani had the least frequency and colonization percentage. Isolates of fungi were identified according to morphological characteristics, cultural, and molecular biology based on internal-transcribed spacer-1 (ITS1) sequencing and conserved in GenBank with accession numbers from (ON037457.1 to ON037474.1). Fungal isolates were varied for causes of root rot on grapevine plants and their reduction effect on the characteristics of growth of flame-seedless cultivar. Fusarium solani isolate (ON037462.1) was the most pathogenic isolate that caused (100%) of root rot, high disease severity, and highly significant reduced grapevine plant growth characteristics followed by isolates of Lasiodiplodia theobromae (ON037474.1). In this respect, a new isolate of fungal species was hosting grapevine plants, i.e., two isolates each of F. chlamydosporum, F. brachygibbosum, one isolate of F. ipomoeae, and one isolate of L. exigua are the new causal pathogens of root-rot disease on grapevine as the first report in Egypt.

Characterizing the Pathogenicity and Mycotoxin Production Capacity of Fusarium spp. Causing Root Rot of Angelica sinensis in China.

Root rot is a very destructive soil-borne disease, which severely affects the quality and yield of Angelica sinensis in major planting areas of Gansu Province, China. Twelve Fusarium strains were identified from root rot tissue and infected soil in the field by comparing each isolate strain internal transcribed spacer, translation elongation factor 1-α sequence and RNA polymerase second largest subunit gene with the sequences of known fungal species in the NCBI database. Of these isolates, four were F. acuminatum, followed by three F. solani, two F. oxysporum, and one each of F. equiseti, F. redolens, and F. avenaceum. Under greenhouse conditions, pathogenicity testing experiment was carried out using five strains: two F. acuminatum, one F. solani, one F. oxysporum, and one F. equiseti. Among them, the incidence of F. acuminatum-induced root rot on A. sinensis was 100%; hence, it was the most aggressive. Liquid chromatography was used to show that F. acuminatum could produce neosolaniol (NEO), deoxynivalenol, and T-2 toxins. Of these, the level of NEO produced by F. acuminatum was high compared with the other two toxins. By isolating Fusarium spp. and characterizing their toxin-producing capacity, this work provides new information for effectively preventing and controlling A. sinensis root rot in the field as well as improving the quality of its medicinal materials.

End-to-end multimodal 3D imaging and machine learning workflow for non-destructive phenotyping of grapevine trunk internal structure

Quantifying healthy and degraded inner tissues in plants is of great interest in agronomy, for example, to assess plant health and quality and monitor physiological traits or diseases. However, detecting functional and degraded plant tissues in-vivo without harming the plant is extremely challenging. New solutions are needed in ligneous and perennial species, for which the sustainability of plantations is crucial. To tackle this challenge, we developed a novel approach based on multimodal 3D imaging and artificial intelligence-based image processing that allowed a non-destructive diagnosis of inner tissues in living plants. The method was successfully applied to the grapevine (Vitis vinifera L.). Vineyard’s sustainability is threatened by trunk diseases, while the sanitary status of vines cannot be ascertained without injuring the plants. By combining MRI and X-ray CT 3D imaging with an automatic voxel classification, we could discriminate intact, degraded, and white rot tissues with a mean global accuracy of over 91%. Each imaging modality contribution to tissue detection was evaluated, and we identified quantitative structural and physiological markers characterizing wood degradation steps. The combined study of inner tissue distribution versus external foliar symptom history demonstrated that white rot and intact tissue contents are key-measurements in evaluating vines’ sanitary status. We finally proposed a model for an accurate trunk disease diagnosis in grapevine. This work opens new routes for precision agriculture and in-situ monitoring of tissue quality and plant health across plant species.

Novel Helicoma and Neohelicosporium (Tubeufiaceae, Tubeufiales) species and two new host records of Helicoma on tropical palms (Arecaceae) from China.

Asexual species of Tubeufiaceae are characterised as helicosporous hyphomycetes and are abundantly discovered in tropical and subtropical regions. The present study collected helicosporous fungal samples from rotting tissues of Caryotamitis, Elaeisguineensis and E.oleifera in Xishuangbanna, Yunan Province, China. Fungal isolates were identified, based on the morphological characteristics and multi-gene phylogeny with DNA sequence data of the internal transcribed spacer (ITS), part of the large subunit nuclear rRNA gene (LSU), translation elongation factor 1-alpha gene (tef 1-α) and RNA polymerase II second largest subunit gene (rpb2). Herein, we introduce three new species viz. Helicomaoleifera, Neohelicosporiumguineensis and N.xishuangbannaensis. In addition, we introduce two new host records of Helicomaguttulatum and H.rufum on Caryotamitis. The illustrations of all identified species, detailed descriptions and in-depth phylogenetic analyses are provided. Our results add new knowledge of fungal species associated with palm hosts in southern China. Moreover, our data will contribute to the biodiversity of fungi in tropical China.

Two new species and a new host record of Pleosporales (Dothideomycetes) from palm (Arecaceae) in Guangdong Province, China

ABSTRACT Pleosporales is the largest order of Ascomycota which is distributed worldwide. Among the wide range of hosts, Pleosporales species are frequently isolated from palms. During the surveys of palm fungi from 2020 to 2021, rotting tissues of Licuala peltata var. sumawongii, Phoenix roebelenii and Trachycarpus fortunei were collected in Guangzhou, Guangdong Province, China. Fungal isolates were identified based on morphological characteristics and multi-gene phylogenetic analyses. In the present study, we introduce two new species viz. Byssosphaeria guangdongense and Paraconiothyrium guangdongensis. In addition, we introduce a new host record for Trachycarpus fortunei. Photo plates, detailed descriptions, and in-depth phylogenetic analysis results are provided for all identified taxa. Our findings expand the understanding of the fungi that are associated with palm hosts. Moreover, our data will contribute to the knowledge of the biodiversity of South China.

Fusarium equiseti as the pathogen causing stem rot disease of red-fleshed dragon fruit (Hylocereus polyrhizus) in India

Dragon fruit (red skin and red fleshed, Hylocereus polyrhizus), newly introduced cactus plant in Mizoram, India, exhibited stem rot (35.38%) disease in established orchards during 2017–2018. Recorded symptoms included small red-brown spots encircled with yellowish halos. The size of the spots gradually enlarged and later the tissues turned completely watery and rotten. Fusarium species were consistently isolated from the stem rot tissues of red-fleshed dragon fruit. The pathogen associated with the stem rot disease was identified as Fusarium equiseti based on the morphological characters (production of abundant macroconidia, single-celled microconidia, and solitary and intercalary chlamydospores) and multigene phylogenetic analysis of internal transcribed spacer of ribosomal DNA (ITS-rDNA), translation elongation factor 1α (TEF-1) and beta-tubulin (TUB2). The phylogenetic trees showed that all isolates belonged to the F. equiseti, a member of Fusarium incarnatum-equiseti species complex (FIESC). Four isolates were tested for pathogenicity that produced the symptoms of stem rots (up to 57.64% severity) at 15 days after inoculation. Koch's postulate was further established by re-isolating the F. equiseti from artificially infected plants. To our best knowledge, this is the first report of F. equiseti as a pathogen causing the stem rot of dragon fruit (red-fleshed) in India.

Fusarium verticillioides Causing Root and Stem Rot in Papaya (Carica papaya) in Mexico.

Mexico is the fifth largest producer of papaya in the world with an estimated production of 1, 134, 753 metric tons per year (FAOSTAT 2022). In February 2022, in the center zone of Sinaloa State (Mexico), in a seedling-producing greenhouse, papaya seedlings were observed with an incidence (20%) of root and stem rot and necrotic tissue. Symptomatic tissues were collected from 10 papaya plants, which were cut into small pieces and surface sterilized sequentially with 70% alcohol for 20 s and 1% sodium hypochlorite for 2 min, dried, placed on potato dextrose agar (PDA), and incubated at 26°C in darkness for 5 days. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing and morphologically characterized on PDA and carnation leaf agar (CLA) media. On PDA, the colonies produced abundant white aerial mycelium, and the center of old cultures was yellow pigmentation (Leslie and Summerell 2006). From 10-day-old cultures grown on CLA medium, macroconidia were slightly curved, which showed zero to three septa, with some slightly sharp apices, and basal cells with notches, the measurements were from 22.53 to 48.94 x 6.9 to 13.73 µm (n= 50). The microconidia were presented in abundant chains of microconidia. The microconidia presented thin walls, oval and hyaline in shape, forming long chains, measuring 10.4 to 14.25 x 2.4 to 6.8 µm (n= 50). Chlamydospores were not observed. The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced from isolate FVTPPYCULSIN (GenBank accession no. OM966892). Maximum likelihood analysis was carried out using the EF1-α sequence (OM966892) and other species from the genus Fusarium. Phylogenetic analysis revealed that the isolate was Fusarium verticillioides (100% bootstrap). Furthermore, the isolate FVTPPYCULSIN was 100 % similar with other reported Fusarium verticillioides sequence (GenBank accession nos. MN657268) (Dharanendra et al. 2019). Pathogenicity tests were performed on 60-day-old papaya plants (cultivar Maradol) grown on autoclaved sandy loam soil mix. Ten plants per isolate (n = 10) were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate per plant. The suspension was obtained by collecting the spores of each isolate grown on PDA with 10 ml of an isotonic saline solution. Ten noninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the greenhouse was observed on the papaya plants. No symptoms were observed on noninoculated control plants after 60 days. The pathogen was reisolated from the necrotic tissue from all inoculated plants and was identified again as Fusarium verticillioides by sequencing the partial EF1-α gene again and based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch's postulates. The molecular identification was confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. The isolate FVTPPYCULSIN was deposited in the fungal collection of the Faculty of Agronomy of the Autonomous University of Sinaloa. To our knowledge, this is the first report of root and stem rot of papaya caused by F. verticillioides. Papaya is an important fruit crop in Mexico, and the occurrence of this disease needs to be taken into account in papaya production.

First report of root rot caused by Fusarium commune on American ginseng (Panax quinquefolium L.) in China.

American ginseng (Panax quinquefolium L.) is one of the most valuable herb crops because of its unique pharmacological effects. In 2019, American ginseng plants withered and root rot with incidences of 20-45% were observed in about 70000m2 of ginseng production field located in mountainous valley of Benxi city (41º23'32" N, 124º04'27" E), Liaoning Province in China. Disease symptoms included chlorotic leaves with dark brown discoloration extending gradually from the basal to the apical part of the leaves. Water-soaked, irregular lesions appeared on the surface of roots and rotten at later stage. Twenty-five symptomatic roots were surface-sterilized by immersion in 2% sodium hypochlorite (NaOCl) for 3 min, followed by rinsing three times in sterilized water. The sections healthy tissues bordered rotten tissues, i.e. the leading edge, were cut into 4-5 mm pieces with a sterile scalpel and 4 pieces were placed on each PDA plate. After 5 days incubation at 26°C, total of 68 single spores were obtained from the colonies with an inoculation needle under stereomicroscope. Colonies from single conidia were white to greyish white, densely floccose to fluffy, and the reverse grayish yellow with dull violet pigmentation. Single-celled and ovoid microconidia in false heads were borne on aerial monophialidic or polyphialidic conidiophores on Carnation Leaf Agar (CLA) media, and measured 5.0 -14.5 × 3.0 -4.8 μm (n=25). Macroconidia were two to four septa, slightly curved, apical and basal cells were also curved, and they measured 22.5 - 45.5 × 4.5 - 6.3 μm (n=25). Chlamydospores were singly or in pairs, circular or subcircular, smooth, and measuring 5 - 10.5 μm (n=25) in diameter. Morphologically, the isolates were identified as Fusarium commune (Skovgaard et al. 2003; Leslie and Summerell 2006 ). To confirm the identity, the rDNA partial translation elongation factor1 alpha (TEF-a) gene and the internal transcribed spacer (ITS) region of ten isolates were amplified and sequenced (O'Donnell et al. 2015; White et al. 1990). Identical sequences were obtained, and one representative sequence of isolate BGL68 was submitted to GenBank. BLASTn analysis of both the TEF-α (MW589548) and the ITS (MW584396) sequences, revealed 100% and 99.46 % sequence identity to F. commune MZ416741 and KU341322, respectively. The pathogenicity test was conducted under greenhouse conditions. The surface of healthy 2-year-old American ginseng roots was washed and disinfested in 2% NaOCl for 3 min before rinsing in sterilized water. Twenty roots were wounded with a toothpick, resulting in tiny perforations (1.0 × 1.0×3.0 mm), 3 perforations were wounded on each root. Inoculums was prepared from the culture of isolate BGL68 incubate in potato dextrose broth (PD) for 5 days at 26°C,140 rpm. Ten wounded roots were immersed in a conidial suspension (2 × 105 conidia/ml) for four hours in a plastic bucket, and planted in five containers (two roots per container) filled with sterile soil. Another ten wounded roots were immersed in sterilized distilled water and planted in five containers as controls. The containers were incubated for four weeks in a greenhouse at temperature between 23°C and 26°C, under a 12-hr light and dark regime, and irrigate with sterile water every 4 days. Three weeks after inoculation, all inoculated plants exhibited chlorotic leaves, wilting and root rot. The taproot and the fibrous roots showed brown to black root rot and no symptoms in non-inoculated controls. The fungus was reisolated from the inoculated plants, but not from any of the control plants. The experiment was repeated two times with similar results. This is the first report of root rot caused by F. commune on American ginseng in China. The disease might bring a threat to this ginseng production and should be implemented effective control measures to reduce losses.

First Report of Fusarium solani and Fusarium oxysporum Causing Basal Stem Rot on Celery in China.

Celery (Apium graveolens L.) is an economically important vegetable crop in China. In recent years, celery has been widely planted in Yuzhong county, Gansu province. From 11 April to 24 May, 2019-2021, basal stem rot of celery was observed with incidences up to 15% in the Yuzhong region (35°49'N, 104°16'E, 1865 m a.s.l.), which caused serious economic losses to local farmers. Typical symptoms of the disease included wilting and darkening of the basal stem, leading to plant death. To identify the cause of the disease, small pieces (5mm×5mm) of the margin of asymptomatic and rotting basal stem tissues were sterilized with 70% ethanol for 30 s and 3% NaClO for 5 min, then placed on potato dextrose agar (PDA) plates, and incubated at 25℃ (Zhao et al. 2021). Twenty-seven single-conidium isolates with morphological characteristics similar to Fusarium spp. were obtained (Ma et al. 2022), which displayed two kinds of colony morphology. On PDA, seven isolates developed white, fluffy aerial mycelium and twenty isolates developed light pink abundant aerial mycelium. Isolate F5 and F55 from each distinct morphological group were cultured on PDA and synthetic low nutrient agar (SNA) for pathogenicity tests, morphological and molecular identification. The macroconidia (18.3 to 29.6 × 3.6 to 5.3 μm, n=50) with 1 to 2 septa and microconidia (7.5 to 11.6 × 2.6 to 3.5 μm, n=50) with 0 to 1 septum were observed in F5. For F55, the size of macroconidia was 14.2 to 19.5 × 3.3 to 4.2 μm (n = 50) with 1 to 2 septa; Microconidia were mostly 0 to 1 septum and measured 7.3 to 12.8 × 2.2 to 4.2 μm (n = 50). To confirm the identity of the isolates, the internal transcribed spacer region (ITS) and the translation elongation factor-1 alpha (TEF-1α) gene were amplified using primers ITS1/ITS4 and EF-1/EF-2 (Uwaremwe et al. 2020), respectively. The sequence similarities of isolate F5 (GenBank No. OL616048 and OP186480) and F55 (GenBank No. OL616049 and OP186481) with the corresponding sequences of F. solani (MT447508 and MN650097) and F. oxysporum (MG461555 and OQ632904) ranged from 99.22% to 100.00%, with matching base pairs of 531/532, 416/416, 511/515 and 394/395, respectively. The voucher samples were deposited in the sample center of Northwest Institute of Ecological Environment and Resources, Chinese Academy of Sciences. Morphological and molecular results confirmed the species of F5 and F55 as F. solani and F. oxysporum, respectively. A pathogenicity test was conducted under greenhouse conditions (19-31°C, avg. 26°C). Conidial suspension (105 spores/mL) of isolates F5 and F55 were poured onto the basal stems of healthy celery seedlings at the age of 1 month and mock-inoculated control treatments with sterile water. Ten plants were inoculated for each treatment. After 21 days, all plants inoculated with both fungal isolates developed symptoms similar to those observed in the field, whereas the mock-inoculated plants remained healthy. The pathogen was successfully reisolated from the inoculated symptomatic plants onto PDA medium and had morphology as described above, confirming Koch's postulates. F. solani and F. oxysporum have been reported to infect many plant species, including carrot (Zhang et al. 2014) and Angelica sinensis (Liu et al. 2022). To our knowledge, this is the first report that F. solani and F. oxysporum cause basal stem rot on celery in China. The identification of pathogens of the observed basal stem rot on celery provides a clear target for the prevention and management of this disease.

Nano-metals forming bacteria in Egypt. II. Efficacy towards biomolecules, ultrastructure, growth parameters, and eco-friendly therapeutic of soft rot/blackleg genera

The nanoparticles (NPs) formed by Enterococcus thailandicus, Pseudomonas putida, Marinobacter hydrocarbonoclasticus, and P. geniculate were tested against soft rot/blackleg genera. The effects of NPs recorded on bacterial DNA, proteins, and carbohydrates concentration of Pectobacterium carotovorum subsp. carotovorum, Enterobacter cloacae (soft rot), and Dickeya solani (soft rot/blackleg). Treated cells showed degradation in isolated DNA, decreased proteins and carbohydrates concentration compared with untreated cells. Using Scanning Electron Microscope (SEM), the treated cells showed collapsed and small pits in the cell wall. Using Transmission Electron Microscope (TEM), internal changes showed penetration of NPs inside the tested bacterial cells, the appearance of periplasmic space, formation of vacuoles, and condensation of cytoplasm. Disease severity ex vivo of potato tuber infected with tested genera demonstrated that NPs treatment didn’t show any rotted tissue compared with untreated. The ability to uptake and accumulate FeNPs from the soil in potato (Solanum tuberosum) seedlings; Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) was used. It recorded an increase in iron content of treated potato (Solanum tuberosum) seedlings with NPs, compared with untreated. FeNPs can be used to control soft rot/blackleg diseases, instead of copper pesticides. It could be a new, approach for disease management and increase the plant’s nutritional value.

First Report of root rot Caused by Fusarium solani and F. breve on kiwifruit in China.

China is considered as the main producer of kiwifruit (Actinidia spp.) in the world. During 2020-2021, root rot (~8000 plants, ~5% disease incidence) of 3-year-old kiwifruit (cv. Xuxiang) was observed in Lujiang County (117°24'E, 31°15'N), Anhui, China. This disease usually occurred in fields with poor drainage in hot and humid summers. Symptoms started on leaves showing dehydration and curling, the last root of diseased plant turned black and died. Dig out the skin on rotten root was cracking and flaking and white mycelium covered on surface. Twenty rotten tissues from ten plants were cut and surface disinfected with 1% NaOCl for 5 min, rinsed in sterile water, and cultured on potato dextrose agar (PDA) at 25 ± 2°C in the dark. Fifteen fungal isolates were obtained. The first type (KWRR1, 3-10) was cotton-like, reverse with white outer margin, and light brown inner region on PDA. The second type (KWRR2, 11-15) was cotton-like on PDA but appeared pale yellow in reverse. On oatmeal agar, the KWRR1 colony was flat with little aerial hyphae and was red, while KWRR2 was hyaline. On carnation leaf agar (CLA), microconidia of the KWRR1 and KWRR2 isolates were reniform, fusiform or oblong, 0-1 septate, and measuring 1.9-4.3×8.4-15.7 µm and 3.0-3.8×8.2-16.7 µm, respectively (n=50). The macroconidia of KWRR1 were straight or moderately curved, 3-5 septa (2.7-4.6×21.5-52.6 µm in size, n=50). For KWRR2, the macroconidia were straight or slightly curved and with 3-4 septate, 4.1-4.8×26.1-30.8 µm (n=50). Chlamydospores of the KWRR1 and KWRR2 isolates were 1-2 celled, irregular globose, measuring 4.5-8.5 µm and 7.6-9.0 µm diam, respectively (n=50). To identify the isolates, four DNA fragments (RPB1, RPB2, ITS and TEF-1α) were amplified and sequenced from all isolates (O'Donnell, et al. 2012; White et al. 1990; O'Donnell et al. 2022). BLAST analysis of the RPB1, RPB2, ITS and TEF-1α sequences of the KWRR1 isolates (OL474057, OL474055, OL468550, OP382187) showed highest identity with F. solani (NRRL66304; MW218134, KT313623, KT313633, KT313611) at 98.2%-99.8%, while KWRR2 (OL505579, OL474056, OL468551, OP382188) showed that their homology with F. breve (NRRL28009; HM347149, EF470136, DQ094351, DQ246869) at 98.2%-99.4%. F. solani and F. breve belong to clade 3 of the F. solani species complex (FSSC) (Geiser et al. 2021). Phylogenetic analysis based on RPB2, ITS and TEF-1α sequences with MEGA7 software (Šišić et al. 2018), placed the KWRR1 sequences with F. solani (FSSC5), while there of KWRR2 nested with F. breve (FSSC15). One-year-old seedlings (n=6) of 'Xu Xiang', growing in a greenhouse (at 28℃, relative humidity 80%), were inoculated by drenching the soil with a conidial suspension with one of the two isolates (30 ml, 106 conidia/ml). Control plants (n=6) were inoculated with sterilized water and the pathogenicity assay was repeated three times. One month post-inoculation, the leaves of inoculated plants became chlorotic, wilted and died, whereas the controls were disease-free. F. solani and F. breve were successfully reisolated from diseased samples (n=6) and verified based on morphology and sequencing as described above, fulfilling Koch's postulates. Members of the FSSC cause root rot on many hosts (Coleman. 2016; Schroers et al. 2016), but this is the first report of F. solani and F. breve causing root rot disease on kiwifruit in China. The result will serve as the foundation for management of root rot of kiwifruit.

Aetiology and molecular characterization of the pathogens associated with soft rot disease of Aloe vera (L.) Burm. f.

Soft rot is a devastating disease of aloe and the infected plants developed symptoms of rotting tissues from collar region upwards. Aloe plants artificially inoculated with a Dickeya spp. (reported earlier as the causal agent) at the root zone but did not develop the disease symptoms, eliminating the possibility of pathogen to entering through roots. Besides, a Fusarium spp. was repeatedly isolated from collar regions of the naturally infected plants. Further, it was established that lesions produced due to fungal infection predisposed the subsequent infection of the bacterial pathogen. Sequencing results and phylogenetic analysis based on three partial genes of bacteria (dnaX, icdA and mdh) and fungus (ITS, TEF-1α and RPB-2) confirmed the identity of pathogens as Dickeya zeae and Fusarium falciforme, respectively. An artificial inoculation technique was developed for quick screening of aloe germplasm for resistance of bacterium. Among 40 accessions screened, none was found resistant, however, F. falciforme failed to produce lesion on two accessions (Guj4 and Raj3), consequently making them resistant to soft rot disease upon combined inoculation with both the pathogens. Besides the genetic constituent, rapid rotting was observed at 35 °C but not at and below the 15 °C temperature. In planta, the bacterium concentration increased gradually with the rise of incubation temperature between 15 and 35 °C. The present study suggests possible management aspects of the problem through (i) exploiting host resistance and (ii) escaping post–harvest decay by storing and transporting aloe leaves at temperatures ≤ 15 °C and (iii) avoidance of water stagnation in field.

Exploitation of a Prosprosthetic Device for Dental Piling

Objective: In order to solve the effective problem of removing the rotten material in the root canal, and can adapt to the various continuous surgical procedures in the root canal surgery. Methods: When it is necessary to clean the rotten tissue, place the device above the teeth after drilling, insert the inner tube into the hole and extend it to the required depth, and then further adjust the depth of the inner tube in the hole by adjusting the position of the outer tube of the driving mechanism. The driving mechanism is started so that the driving mechanism drives the elastic hollow part to rotate. Then, a liquid with a certain consistency is filled into the first cyst through a pipe so that the first cyst can be effectively propulsive and closely adhered to the corrupt tissue, while the inner tube is continuously pulled upward. The second capsule is continuously filled with liquid through the pipe, so that the second capsule can contact the tissue after cutting and fully contact the surface of the tissue after cutting and complete the sealing. In jecting the clean water into the hole on the teeth. The teeth can be filled through the cavity formed between the elastic hollow and the pipe by pouring the tooth filling material into the hole of the treated tooth. Results: The use of the device can adapt to a variety of continuous operation processes in root canal surgery, the use is more simple and convenient, and the operation is more efficient. Conclusion: The use of this device can adapt to a variety of continuous operation procedures in root canal surgery, which is simpler and more convenient to use, and more efficient to operate.

First Report of Penicillium oxalicum Causing Postharvest Fruit Rot on Citrus in China.

Ougan (Citrus reticulata cv. Suavissima) is a distinct citrus cultivar local to Zhejiang province, China. (Guo et al. 2021). In November 2021, an unknown postharvest fruit rot was observed in the Sanyang wetland Ougan planting area, Wenzhou City of Zhejiang Province (27.96 °N, 120.69 °E). About 3% of diseased fruits with similar fruit rot symptoms were observed in 900 mandarin fruits from four commercial storages. Initially, the symptoms appeared as light brown lesions that turned deep brown as the lesions expanded. To identify the pathogen, segments (5 mm2) from margins of rotted tissue were excised from 5 symptomatic fruits, surface disinfested twice with 75% ethanol, rinsed three times with sterilized water, placed aseptically onto potato dextrose agar (PDA) medium and incubated for 7 days at 25℃ in darkness for identification. Five fungal isolates with the same morphology were obtained using the single spore method (Leslie and Summerell 2006). Initially, the fungus produced fluffy and white aerial mycelium that eventually turned green on PDA medium after 3 days. Conidiophores were broom-shaped (17.5 ± 2.5 μm) (n=50). Conidia were unicellular and ellipsoid (3.5 to 5.0 ×2.5 to 4.0 μm) (n=50). These morphological characteristics were consistent with Penicillium species (Wu et al. 2022). WZU-OG1 was chosen as a representative isolate for further study. For molecular identification, PCR amplification and DNA sequencing were performed using primers ITS1/ITS4 (White et al. 1990), bt2a/bt2b (Glass and Donaldson 1995), and RPB2-5F/RPB2-7R (Liu et al. 1999) to amplify the complete internal transcribed spacer (ITS) region, β-tubulin gene (TUB), and a portion of RNA polymerase second largest subunit (RPB2). The ITS, TUB, and RPB2 gene sequences of isolate WZU-OG1 were deposited in the GenBank database with acc. nos. ON332735, ON428245, and ON524171, respectively. BLASTn analysis respectively showed 561/561 (MH855125), 414/427 (KF296462), and 936/954 (JN121456) matching with Penicillium oxalicum CBS 219.30. A neighbor-joining phylogenetic analysis based on the concatenated nucleotide sequences (ITS, TUB, and RPB2) grouped this isolate in the Penicillium oxalicum species complex clade at 100% bootstrap support. To verify pathogenicity, 20 healthy mandarin fruit of cultivar Sanyang were superficially disinfested with 75% ethanol and then washed with distilled water. A conidial suspension of 1 × 105 conidia/ml from a 5-day-old culture of WZU-OG1 was injected into 10 fruits (10 μL per fruit). An equal number of fruits inoculated with sterile water were used as the negative control. The inoculated fruits were stored in a constant temperature incubator under the conditions of 28 ℃, 90% humidity, and incubated in a 12-h light/12-h dark cycle for 12 days. Symptoms similar to those on the naturally infected fruit began 5 days after inoculation, whereas no symptoms occurred on the controls. The experiment was repeated three times, and similar symptoms were observed in all diseased fruits. Then, the fungus was reisolated from these infected fruits and identified as P. oxalicum by the morphological and molecular methods described above. This is the first report of P. oxalicum causing postharvest mandarin decay and this study will enable us to rapidly diagnose this disease, identify the occurrence of this disease and develop adequate management strategies to control it in China.

Isolation and Identification of Fungi Associated with the Spoilage of Cocoyam Corms

Cocoyam (Colocasia esculenta) is an important tropical root crop grown purposely for its starchy corms. This study was carried out to isolate and identify fungi associated with the spoilage of cocoyam corms. These organisms were isolated from spoilt corms by direct inoculation of the rotten tissues on Sabouraud Dextrose Agar. The fungal isolates were identified using cultural and morphological features such as colony appearance and also through microscopic characteristics. The isolated organisms include Rhizopus stolonifera, Rhizopus arrhizus, Fusarium oxysporum, and Aspergillus niger. Out of the 70 samples that were analyzed, 45 (64%) had Rhizopus stolonifera, 36 (51%) had Fusarium oxysporum, 52 (74%) had Rhizopus arrhizus while 61 (87%) had Aspergillus niger. A pathogenicity test was carried out on healthy corms using isolated microorganisms. The result showed that the organisms were responsible for the deterioration of the corms. These organisms may have infected these corms through contaminated working tools during harvest or improper storage by farmers. Corms are always infected by molds if not properly handled or stored. Hence, proper care should be taken while harvesting, handling, or storing these corms to avoid being infected by these pathogenic organisms since they can be detrimental to human health. It can also cause huge economic losses to farmers.

First report of Botryosphaeria dothidea as the causal agent of a new fruit rot disease of pepper in China.

Pepper is an important and widely cultivated economic vegetable in the world (Yin et al., 2021). In June 2021, approximately 25% to 33.3% of the pepper plants had rot disease symptoms in Zhuanghang Comprehensive Experimental Base (30.894829 °N, 121.391374 °E), Fengxian district, Shanghai city, China. Water-soaked spots appeared on fruits that increased in size and leading to smelly fruit decay. To isolate the pathogen, three pepper samples with severe symptoms were collected. The samples were surface disinfected with 70% ethanol for 30 sec, 10% chlorine bleach for 10 min, rinsing with sterile water for three times and the rot tissues were cut and dried on sterile filter paper. The dried paper was later placed on potato dextrose agar (PDA) medium and incubated at 28°C (Tang et al., 2021). After 2-3 days, four types of colonies with different colony appearances were observed, in which only one can induce fruit rot phenotype (data not shown). Four isolates were cultured for molecular identification in each type. ITS1/ITS4, T1/βt-2b and EF1-526F/EF1-1567R primers were used to amplify the internal transcribed spacer region (ITS), the beta-tubulin (TUB2) and the translation elongation factor I alpha (EF1-α) genes, respectively (Chen et al., 2018) and corresponding sequences from the isolates were analyzed with BLAST. Sequences of the isolate which can induce pepper decay were submitted to GenBank under the accession numbers of OM663701 (ITS), OM720127 (TUB2) and OM720128 (EF1-α). The results showed that the pathogen had 99% sequence homology to most strains of Botryosphaeria dothidea (B. dothidea) and displayed the highest sequence similarity to strain LBSX-1 (ITS: KF55123), strain JGT01 (TUB2: MW202404) and isolate CZA (EF1-α: MN025271). Based on molecular characterization, the isolate was identified as B. dothidea isolate SH01. A phylogenetic tree was constructed using Maximum Parsimony (MP) methods by MEGA7, and showed that SH01 was closely related to isolate CMW9075. To confirm the pathogenicity, five healthy pepper fruits were surface sterilized, 500μl of conidial suspension (1×103 conidia/ml) were injected into pepper (sterilized distilled water as control). Six days post inoculation (dpi), fruit rot symptoms appeared and the pepper decayed at 12 dpi. Four days post inoculation with mycelium plugs (from a 4-day-old culture on PDA, PDA plugs as control), hyphae were observed in the inoculation site and B. dothidea was re-isolated from the symptomatic areas, thus fulfilling Koch's postulates (Back et al., 2021, Chen et al., 2020). The pepper rotted severely at 7 dpi. The colonies of SH01 were pale to white and gradually turned into gray in 4-6 days. Conidia of the pathogen were unicellular, aseptate, hyaline and fusiform to fusoid, with dimensions of 19.7-23.5 μm × 3.8-5.2 μm (average = 21.9 μm × 4.8 μm, n = 50). Hyphae were transparent, branched and composed of multiple cells. The characteristic was consistent with the descriptions of B. dothidea (Vasic et al., 2013). B. dothidea belongs to Botryosphaeriaceae, causing widespread diseases in many plant species, commonly associated with cankers and dieback of woody plants and economic crops, such as plumcot trees (Back et al., 2021), eucalyptus (Yu et al., 2009) and soybeans (Chen et al., 2020) in China and Korea. Our findings reported for the first time that B. dothidea (SH01) can induce the pepper rot disease and future work on its pathogenesis may provide strategies for disease control against this fungus.

Isolation of the Main Pathogens Causing Postharvest Disease in Fresh Angelica sinensis during Different Storage Stages and Impacts of Ozone Treatment on Disease Development and Mycotoxin Production.

Angelica sinensis, a Chinese herbal medicine, is susceptible to molds during storage, reducing its quality, and even generating mycotoxins with toxic effects on human health. Fresh A. sinensis was harvested from Min County of Gansu Province in China and kept at room temperature. Naturally occurring symptoms were observed during different storage stages. Molds were isolated and identified from the diseased A. sinensis using morphological and molecular biology methods. The impact of ozone treatment on postharvest disease development and mycotoxin production was investigated. The results indicated that A. sinensis decay began on day 7 of storage and progressed thereafter. Nine mold species were isolated and characterized: day 7, two Mucormycetes; day 14, Clonostachys rosea; day 21, two Penicillium species and Aspergillus versicolor; day 28, Alternaria alternata and Trichoderma atroviride; and day 49, Fusarium solani. Ozone treatment markedly inhibited the development of postharvest disease and the mycotoxin production (such as, patulin, 15-acetyl-deoxynivalenol, and sterigmatocystin) in the rotten tissue of A. sinensis inoculated with the nine isolates.

Detection and classification of volatile compounds emitted by three fungi-infected citrus fruit using gas chromatography-mass spectrometry

Citrus fruit produced some characteristic volatile compounds when infected by fungi compared with the healthy fruit. In the present study, volatile metabolites of postharvest citrus fruit with three different diseases including stem-end rot, blue mold and green mold were detected. Multivariate analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to classify the volatile compounds between the infected and non-infected citrus fruit. The results indicated that volatile compounds of unrotten, unrotten-rotten junction, and rotten tissues were successfully classified. Importantly, eight volatile compounds as biomarkers for stem-end rot and one biomarker for green mold of citrus were screened to discriminate the infected citrus fruit. This study offers the application potential of odor profiling of volatile compounds for detecting the fungi infection in postharvest citrus fruit.