First report of Wilt on Lentil (Len culinaris Medik.) caused by Fusarium redolens in Tunisia

Lentil (Lens culinaris Medik.) is widely grown in arid and semi-arid regions of Tunisia. Low yields are often attributed to it being grown on marginal growing land. Since 2016, symptoms of wilt including yellowing and discoloration of the stem and root tissues were observed in lentils in several region of Tunisia. The annual mean incidence of infected plants ranged from 10% to 15%. In 2019-2020 growing seasons, symptomatic adult plants were randomly sampled from two fields located in south Tunisia (33°37’N; 11°4’E; N and 33°33’N; 11°2’E), and one field located in north west Tunisia (36°7’N; 8°43’E). Pieces were cut from roots and stem, surface sterilized, then plated on ¼ strength Potato Dextrose Agar (PDA) + 100 mg L-1 streptomycin sulfate (Burgess et al., 1994). Cultures were incubated for 5-6 days. Nine colonies with floccose mycelia, spare or abundant and white to violet color, morphologically similar to Fusarium redolens according to Leslie & Summerell (2006) were isolated from both roots and stems. They were single-spored (Burgess et al., 1994). Microconidia were formed in false heads on short monophialides. They were oval to elliptical or reniform and were 0-1 septate. Three-septate macroconidia with short apical cells were also observed. The strains were also deposited in the microbial ITEM Collection of Institute of Sciences of Food Production. Extraction of genomic DNA of Fusarium sp. strains was carried out according to Wizard® Magnetic DNA Purification System (Promega, Fitchburg, WI, USA). Molecular identification was carried out based on translation elongation factor (TEF) gene sequencing, as described in Fallahi et al. (2019). The TEF sequences were searched on GenBank database by using the Basic Local Alignment Tool (BLAST). The sequences of four strains (Z2P6, Z2P7, Z3P2 and Z3P5) on a total of nine, recovered from lentil roots from the two fields of south Tunisia showed 100% homology with TEF sequences of the epitype culture of F. redolens NRRL25600 (accession number MT409453) (Balmas et al. 2010; Gargouri et al. 2020). Sequences of the strains were submitted to GenBank with accession numbers MW393853, MW393854, MW393856 and MW393857. Pathogenicity of the four strains of F. redolens was evaluated on Kef lentil variety (Kharrat et al. 2007). Inoculum was produced on sterilized oat colonized with each strain. The colonized grains were air-dried on filter paper, ground in a laboratory mill, mixed at 10 % to soil (10 g of each isolate inoculum for 100 g of disinfected soil substrate) and potted. Three germinated lentil seeds were placed in each pot and irrigated periodically. The test was replicated four times. After 21 days, 60% (33-100%) of the plants inoculated with the four F. redolens strains showed symptoms of wilting, yellowing and rotting of roots and 17% died when inoculated by Z2P6 and Z2P7 strains. Non-inoculated plants showed no symptoms. F. redolens was isolated from 100% of the inoculated plants roots. This is the first report of F. redolens as a pathogen on lentil in Tunisia. This species has also been associated with lentil wilting in other regions of the world including Italy (Riccioni et al. 2008), Canada (Taheri et al. 2011) and Pakistan (Rafique et al. 2020). F. redolens was previously reported from wilted chickpea crops in Tunisia (Bouhadida et al. 2017). These findings are important for the Tunisian national legumes program and call for larger surveys to better understand the biology and ecology of this species and to prevent from disease spreading.

Production of amylases by some aspergillus and fusarium species isolated from waste corncobs in Keffi, Nigeria

Amylases are important industrial enzymes that have wide applications ranging from conversion of starch to sugar syrups, to the production of cyclodextrins for the pharmaceutical industry. This investigation aimed at production of amylases using Aspergillus and Fusarium species isolated from waste-corncobs in Keffi Nigeria. Standard microbiological methods were employed for isolation and identification of the fungal isolates. The yields of amylases produced by fungi isolates were determined using Spectrometry. The isolation rate of Aspergillus and Fusarium species was high in location A, C and D with 60% and location B with 40%. The percentage occurrence of the isolates demonstrated that Aspergillus carneus was 40%, Aspergillus aculeatus was 60% and Aspergillus flavus was 20% while Fusarium moniliforme was 80% and Fusarium redolens was 40%. The result demonstrated that three species of the fungal isolates Aspergillus aculeatus, Aspergillus carneus and Fusarium moniliforme were found to produce amylases. Aspergillus aculeatus isolated from locations C3, D1 and D2 produced 0.018mg/ml, 0.018mg/ml and 0.016mg/ml amylases respectively. Similarly, Aspergillus carneus isolated from locations A1 and B2 produced 0.021mg/ml and 0.012mg/ml amylases. Fusarium moniliforme isolated from locations A3, C1 and C4 produced 0.010mg/ml, 0.016mg/ml and 0.015mg/ml amylases. Result of effect of (temperature, pH and fermentation time) for production of amylases. Whereas highest amount for amylases produced by Aspergillus aculeatus and F monliforme were produced at 28 OC. pH 5.0 was found to the best optima pH for production of amylases from the fungi studied A. carneus (2.99 mg/ml amylases). The fermentation time showed highest production of amylase by A. carneus and A. aculeatus after 72 hours while F. moniliforme produced at 96hours. The fungi species isolated from soil in keffi can be used for production of amylases.

Evaluation of the efficacy and optimization of indigenous microbial isolate for herbicides (Paraquat Dichloride, Glyphosate, and Glyphosate isopropylamine) degradation

The presence of herbicides in soil is a serious problem for the environment. Studies on degradation of Herbicide (Paraquate dichloride (PD), Rake out (RO) and Gobara (GB)) by bacteria and fungi species isolated from soil environment in Keffi Metropolis Nigeria were carried out. A total of twenty (20) soil samples were collected. The bacteria and fungi were isolated from the soil and identified using standard microbiological methods. The herbicides utilization was determined using Atomic Adsorption Spectrometer. The effect of temperature on utilization of the herbicides by Enterobacter asburiae at 26OC ranges from 1.23±0.11 mg/ml for PD, 1.14±0.29 mg/ml for RO and 0.53±0.86mg/ml for GB, Pseudomonas aeruginosa utilization ranges from 1.45±0.17 mg/ml for PD, 1.17±0.35 mg/ml, for RO 1.12± 0.82mg/ml for GB. Aspergillus flavus ranges from 2.12±0.19 mg/ml for PD, 2.00±0.03 mg/ml for RO and 2.02±0.57 mg/ml for GB, Fusarium redolens were 2.19±0.26 mg/ml for PD, 2.15±0.08 mg/ml for RO and 1.92±0.16 mg/ml for GB. Effect of incubation time on microbial herbicides degradations: for E. asburiae on PD it ranges from day 1 with 0.24±0.37 mg/ml to day 20 with 2.06±0.11 mg/ml. for P. aeruginosa on PD ranges from day 1 with 0.38±0.08 mg/ml to day 20 with 2.39±1.45 mg/ml. The Utilization of herbicides by A. flavus on PD ranges from day 1 with 0.10±0.01 mg/ml to day 20 with 2.29±0.12 mg/ml. for F. redolens in PD, it ranges from day 1 with 0.27±0.08 mg/ml to day 20 with 2.57±0.27mg/ml. The process of degradation of herbicide has become very attractive as it allows for removal of herbicide over a relatively broad range of pH and temperature

Molecular and phylogenic identifications of potential herbicide degrading microorganisms from contaminated farmland in Keffi, Nasarawa State, Nigeria

Background: Over the past years, the continuous use of herbicides has raised increasing concern mainly due to their massive pollution of the environment. To address this problem, the herbicide-degrading microorganism might be very promising. Method: In the present study, a total of twenty (20) soil samples were collected, bacteria and fungi were isolated from the soil and identified using standard microbiological and molecular studies. Results: The results revealed that a total of 2 bacterial (Enterobacter asburiae and Pseudomonas aeruginosa) and 2 fungi (Aspergillus flavus and Fusarium redolens) strains were isolated from the soil samples collected from herbicide contaminated soil in Keffi, Nasarawa State, Nigeria. The E. asburiae and P. aeruginosa had 75.0%, and 100% occurrence while the fungi isolates including the A. flavus and F. redolens had 75.0%, and 100% occurrence respectively. The 16S rDNA and ITS1F analysis confirmed the identity of the bacteria and fungi genus. Phylogenetic analysis suggested the bacteria strains were closely related to Pseudomonas aeruginosa strain CIFRI DTSB1 and Enterobacter sp revealed a close relatedness with Enterobacter asburiae RD-DAROS-04 strain, whereas, the fungi strain revealed a closely relatedness of Fusarium redolens strainTIST190421511. Conclusion: In conclusion, the microbial strains including Pseudomonas aeruginosa strain CIFRI DTSB1, Entrobacter asburiae RD-DAROS-04, and Fusarium redolens strainTIST190421511 were the major microbial habitant of pesticide-contaminated farmland in Keffi, and thus represent the herbicide degrading microorganism in this region. Further studies on the herbicide degrading properties of these microbial strains are underway.

The Whole Genome Sequence of Fusarium redolens strain YP04, a Pathogen that Causes Root Rot of American Ginseng

Fusarium redolens was previously reported as a plant pathogen or an endophyte that is closely related to F. oxysporum, a notoriously significant soil-borne phytopathogen. Subsequent studies demonstrated the unique nature of F. redolens and was considered a distinct species that causes multiple symptoms on multiple hosts. It was recently identified as a pathogen that causes root rot of American ginseng. Currently, few high-quality F. redolens genome sequences exist in the public database. Here, we report the whole genome sequence of F. redolens strain YP04, based on a hybrid assembly long- and short-read sequencing with PacBio and Illumina platforms, respectively. The assembly consists of 40 configs with a total length of 52.8 Mb nuclear genomic DNA and 49.6 kb complete mitochondrial genomic DNA, and encodes a total of 18,985 genes including 18,517 protein-coding genes and 469 RNA genes which were functionally annotated. A total of 4606 proteins were identified in the pathogen host interactions (PHI) database, suggesting they were likely involved in pathogenicity and host-pathogen-host interactions, while 41 secondary metabolite synthesis clusters were predicted and annotated. This is the first high-quality whole genome of F. redolens, providing an important community resource for genome evolution, host-pathogen-host interaction, and secondary metabolite biosynthesis studies.

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal rot caused by Fusarium redolens in China

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal stem rot caused by Fusarium redolens in China Tao Tang1, Fanfan Wang1, Jie Guo1, Xiaoliang Guo1, Yuanyuan Duan1,Jingmao You1* 1 Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi, 445000, China. Duohua huangjing (Polygonatum cyrtonema Hua), a herbal medicine, that is mostly planted in several provinces in China. In April 2020, severe diseases with about 40% seedling losse was found in the Huangjing seedling base in Shiyan city, Hubei province. The symptoms included softening and decay of the roots and stem bases, a progressive yellowing and wilting of leaves, and finally being completely rotted. Small pieces of symptomatic stems (0.5 cm in length) and leaves (0.5 × 0.5 cm in size) were surface sterilized with 75% ethanol for 30 s, followed by 0.1% HgCl2 for 1 min, rinsed three times with sterile water, and then dried with sterilized absorbent paper. The sections were placed on potato dextrose agar (PDA) medium containing 10 µg/ml of ampicillin and incubated at 25°C in the dark. After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Macroconidia were sickle-shaped, 15.8 - 32.3 × 3.1 - 5.6 μm (n = 25), and three to five septate. Microconidia were oval or kidney-shaped, 5.2 - 11.4 × 2.0 - 3.2 μm (n = 25), and zero to one septate. To confirm the identity of the pathogen, molecular identification was performed with strain HJCD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) using ITS1/4 (White et al. 1990) , EF1/EF2 (Taylor et al. 2016), respectively. Following BLAST searches and phylogenetic reconstruction, the ITS region (GenBank MW485770.1) showed 99% identity with those of Fusarium redolens in GenBank (KU350713.1) and the TEF-1α (GenBank MW503930.1) showed 100% identity with F. redolens GenBank (MK922537.1). Pathogenicity tests were performed to fulfill Koch’s postulates. Huangjing seedlings were rinsed with sterile water, wiped clean with sterile absorbent paper, and transferred to a tray covered with wet filter paper to maintain high humidity. The mycelial piugs of F. redolens HJCD1 were inoculated onto the surface of leaves and basal stems. Controls were inoculated with sterile PDA plugs. The inoculated seedlings were sealed with plastic wrap, and then cultivated in a 25 ℃ growth chamber with 16 h of light per day. The pathogen-inoculated plants exhibited etiolation and typical wilt symptoms after 4 days, whereas no symptoms were observed in the control plants. F. redolens was reisolated from the infected tissues, and colony morphology and ITS sequence of re-isolates were same as that of HJCD1. The pathogen has been reported previously in american ginseng in China (Fan et al. 2021), lentil in Pakistan (Rafique et al. 2020), and wild rocket in United Kingdom (Taylor et al. 2019). However, to the best of our knowledge, this is the first report of F. redolens causing seelding basal rot on Duohua huangjing in China. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Taylor, A., et al. 2016. Mol. Plant Pathol. 17:1032. https://doi.org/10.1111/mpp.12346 Fan, S. H., et al. 2021. Plant Dis. https://doi.org/10.1094/PDIS-11-19-2519-PDN Rafique, K., et al. 2020. Plant Dis. 9:104. https://doi.org/10.1094/PDIS-11-19-2519-PDN Taylor, A., et al. 2019. Plant Dis.6:103. https://doi.org/10.1094/PDIS-12-18-2143-PDN Funding: Science Funds for Young Scholar of Hubei Academy of Agricultural Science (grant no. 2020NKYJJ20), National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ03), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R. China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no.2020ZTSJJ6)

First Report of Crown Rot Caused by Fusarium redolens on Wheat in Kazakhstan

Fusarium crown rot, caused by several species within the genus, is a major constraint that results in significant losses in wheat production worldwide. In June 2019, diseased wheat plants with typical symptoms of crown rot, including discoloration on the first two or three internodes of the stem just above the soil line and stunted, dry rotted, and discolored roots were collected in several bread wheat fields during the maturity stage in Almaty, East Kazakhstan, and Karaganda Regions of Kazakhstan. For each field, approximately twenty tillers were randomly sampled. Symptomatic tissues were surface sterilized in 1% NaClO for 2 min, rinsed with sterile distilled water three times, air-dried in a laminar flow hood, and then transferred to Petri dishes containing one-fifth strength potato dextrose agar (PDA). After incubating in the dark at 23°C for 5 days, 79 single-spore isolates showing cultural and microscopic characteristics of Fusarium were obtained on PDA and Spezieller-Nährstoffarmer agar (SNA). Colonies were initially white but later produced a beige to pink diffusible pigment in PDA. Microconidia that formed on aerial monophialides were hyaline, 0 to 1 septum, oval- to kidney-shaped, and measured 4.3 to 10.3 × 1.9 to 3.4 µm (average 7.8 × 2.6 µm), whilst macroconidia were straight to slightly curved, 3 to 5 septate, and measured 18.7 to 38.8 × 2.9 to 6.6 µm (average 29.9 × 4.7 µm), with foot-shaped basal cells on SNA. Chlamydospores were present on PDA. Sequence analysis based on portions of translation elongation factor 1α (TEF1) and the nuclear ribosomal internal transcribed spacer region (ITS rDNA) loci with primers EF1/EF2 (O’Donnell et al. 1998) and ITS1/ITS4 (White et al. 1990) identified 29 of the 79 isolates as Fusarium redolens Wollenw. The sequences of the five representative isolates with 99.85% of similarity to those of F. redolens strains available in GenBank e.g., ITS (MT435063) and TEF1 (GU250584). The TEF1 (accession nos. MW403914-MW403918) and ITS rDNA (accession nos. MW397138-MW397142) sequences of the isolates were deposited in GenBank. The morphological features are consistent with the described features of F. redolens (Leslie and Summerell 2006). To confirm pathogenicity of the five isolates, five pre-germinated seeds of wheat cultivar Seri 82 were placed in a 9-cm-diameter pot filled with a sterile potting mix containing equal volumes of peat, vermiculite, and soil. An approximately 1-cm-diameter 7-day-old mycelial plug of each isolate was individually placed in contact with the seeds. Seeds were covered with the same potting mix, and then the pots were maintained for four weeks in a growth chamber at 23°C with a 12-h photoperiod. The experiment was conducted twice with three replicate 15-cm pots with 5 plants per pot. Controls were inoculated with sterile agar plugs using the same procedure. After four weeks, all the inoculated plants showed stunted growth with brown discoloration in most parts of the crown and roots, whereas no symptoms were observed in the control plants. The mean severity of the disease for each isolate was between 2.1 and 2.7 according to the scale of 1 to 5 described by Gebremariam et al. (2015). The pathogen was reisolated from crowns of diseased plants, but not from asymptomatic control tissues, and identified morphologically based on the methods described above, fulfilling Koch’s postulates. Although several morphological features are shared by F. oxysporum and F. redolens, Baayen et al. (2001) showed that these species could be easily distinguished using molecular data. The pathogen was previously reported as F. redolens associated with crown rot of wheat in Turkey (Gebremariam et al. 2015) and Saskatchewan, Canada (Taheri et al. 2011). The presence of F. redolens causing crown rot is confirmed in the six wheat fields surveyed in Kazakhstan, for the first time. This pathogen may pose a risk for wheat production, and further studies needed to determine the impact on the crop in Kazakhstan.

American Ginseng Root Rot Caused by Fusarium redolens in China

American ginseng (Panax quinquefolium L.) originating from North America is one of important herbal medicine and economic crops . With the increasing market demand, China has become the third producer and the largest consumer country of American ginseng. However, continuous cropping obstacle has become the most serious problem for the production of American ginseng, and the continuous cropping of soils usually lead to accumulations of root fungal pathogens and increasing plant disease occurrence (1), root rot caused by the notorious soil-borne pathogenic fungi, Fusarium spp., results in a significant reduction of yield and quality of American ginseng. Investigation of American ginseng root rot was carried out in Liuba county, Shaanxi province, China from 2017 to 2019. About 20% of over 3-year-old American ginseng showed varied root rot symptoms in newly reclaimed fields, and more than 70% in continuous American ginseng planting fields. Among these root rot diseases, we found one kind of disease which shows symptoms of red leaves in initial stage and yellow or yellow brown lesions at the reed heads or taproots. The lesions mainly appear on the root surface; however, the vascular tissue has no discoloration. The aboveground parts become wilted and died, and the whole root appears dark brown rots. Fifteen Fusarium spp. isolates were obtained by cutting diseased rot roots into 5 × 5 mm2 pieces, disinfecting in 70% ethanol for 1 min, rinsing 2 ~ 3 times in sterile water for 1 min and isolating on PDA medium including 50 μg/mL streptomycin sulfate. All the isolates have identical morphological characteristics. The colony was white with curved and uplifted aerial hyphae in central region. The colony diameter was 48 ~51 mm after 6 days at room temperature. Microconidia were oval to cylindrical shape with 0 to 1 septa, ranged from 6.24 to10.09 μm long; the macroconidia were fusiform to conical with a hooked apical cell and a foot-shaped basal cell, usually 3 to 5 septa, ranged from 31.45 to 42.52 μm long. The chlamydospores were not found under our culture condition. Preliminary data analysis showed that the morphological characteristics of these isolates were consistent with the descriptions of Fusarium redolens (2). To clarify the fungus in the taxonomy , the rDNA internal transcribed spacer (ITS), the translation elongation factor 1 alpha (TEF1-α) and the RNA polymerase II subunit 1 (RPB1) fragments of two randomly selected isolates were amplified and sequenced. The sequences of the corresponding fragments of the two isolates were identical. The blast results in the GenBank and FUSARIUM-ID databases show the isolates belong to F. redolens (3). Previous study indicated F. redolens has an indistinguishable relative, F. hostae (4). Although the ITS sequence (MW331695) cannot provide enough information to distinguish them, the phylogenetic tree combined the sequence of TEF1-α (tempID: 2407237 ) and RPB1 (tempID: 2407229) clearly showed that the isolates are F. redolens. (Fig) The pathogenicity of a representative isolate, YP04, was tested on ginseng taproot by in vivo inoculation experiments with three replications. The taproot surface of 2-year-old healthy ginseng was washed and disinfested with 75% alcohol for 1 min and rinsed with sterile water, and dried. The surface of taproot was injured with sterilized steel needles and immersed in 1 × 106 /ml spore suspension (sterile water for control plants) for 30 min. The treatment and control plants were transplanted in 20 cm diameter flowerpots filled with sterilized humus and cultured in a greenhouse at 18-23°C. Six days after transplanting, the leaves began to turn red. The cortex of ginseng taproot showed yellow brown lesions and the vascular tissue turn to light yellow. Fifteen days after transplanting, the aboveground parts of treatment plants began to wilting and the taproots showed serious rots. no taproot rot was observed in the controls. The pathogen was re-isolated from the diseased taproots successfully. To our knowledge, this is the first report of F. redolens causing root rot of American ginseng in China.