Non-inoculated Control Research Articles

Variation in defense enzymes in GT-2 and GAT-5 varieties of tomato during infection with bacterial wilt disease in Central Gujarat, India

Bacterial wilt caused by Ralstonia solanacearum has been recognized as a serious threat to tomato plants which leads to significant loss in yield in the India and across the world. However, there is less information available about the interaction of R.solanacearum and Tomato bacterial wilt. The aim of the study was to show pathogenicity of bacterial wilt in two tomato varieties one, most susceptible (GT-2) and other tolerant (GAT-5) from Anand Central Gujarat. The enzymatic activity was examined for malondialdehyde (MDA) levels, phenylalanine ammonia lyase (PAL), peroxidase (POX), catalase (CAT), superoxide dismutase (SOD). The tolerant varieties had less disease symptom as compared to the susceptible variety. During bacterial wilt pathogenesis in tomato, causes oxidative burst due to encounter of ROS. Here we report, the total amount of phenol and MDA is significantly higher in the leaves of plants inoculated with R.solanacearum of both varieties over their non-inoculated control plants. The SOD, and PAL activity were lower in tolerant varieties and reduced considerably in susceptible varieties as compared to control plants, apart from PAL, which did not demonstrate a significant decline. When compared to non-inoculated plants, CAT enzyme activity were highest in both susceptible and tolerant varieties. The results demonstrate a rapid and rigorous production of reactive oxygen in plants against bacterial wilt and reveal that both varieties of tomato plants showed significant physiological changes during interaction with the R. Solanacearum.

Fruit Rot Caused by Fusarium sulawesiense on Chili in Guangxi, China

Chilli (Capsicum annuum) is considered as one of the most important vegetables and spice crops throughout the world which is widely cultivated in China. In October 2019, fruit rot symptoms were observed on chilli in Guilin, Guangxi, China (N24°18', E109°45'). Irregular dark green spots initially appeared on the middle or bottom of the fruit, then extended to larger grayish brown lesions and started to rot. In the late stage, the whole fruit dried up after water loss. Three disease samples were collected from three towns of different counties in Guilin where the disease incidence of chilli fruits was 15-30%. The margin of diseased fruits was cut into small pieces (3×3 mm), disinfected with 75% ethanol solution for 10 s, 2% NaOCl for 1 min, and rinsed in sterile distilled water three times. Tissue pieces were separately plated on potato dextrose agar (PDA) and incubated at 25°C for seven days. Fifty-four fungal isolates with similar morphology were consistently recovered from diseased tissues of three fruits, with 100% isolation frequency. Three representatives GC1-1, GC2-1 and PLX1-1 were selected for further analysis. The colonies produced abundant whitish to yellowish aerial mycelium on PDA after 7 days incubation at 25°C in the dark. Macroconidia cultured on carnation leaf agar (CLA) for 7 days were long, hyaline, falcate, with dorsal and ventral lines often gradually wider toward apex, curved apical cell and foot-shaped basal cell, mostly 2 to 5 septa, and ranged from 24.16 to 38.88 × 3.36 to 6.55 μm (average 31.39×4.48 μm), from 19.44 to 28.68 × 3.02 to 4.99 μm (average 23.02×3.89 μm), and from 20.96 to 35.05 × 3.30 to 6.06 μm (average 26.24×4.51 μm) for GC1-1, GC2-1, and PLX1-1, respectively. Microconidia were hyaline, fusoid or ovoid, one-septate or nonseptate, and ranged from 4.61 to 10.14 × 2.61 to 4.77 μm (average 8.13×3.58 μm), from 3.55 to 7.85 × 1.95 to 3.04 μm (average 5.79×2.39 μm), and from 6.75 to 18.48 × 3.05 to 9.07 μm (average 14.32×4.31 μm) for GC1-1, GC2-1, and PLX1-1, respectively. Genomic DNA was extracted from 7-day-old aerial mycelia of these isolates. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM) and partial RNA polymerase second largest subunit (RPB2) were amplified using primers ITS4/ITS1, EF1/EF2, CL1/CL2A, and 5F2/7cR, respectively (White et al. 1990; O'Donnell et al. 2000, 2010). Sequences were deposited in GenBank (ITS: OQ080044-OQ080046; TEF1: OQ101589-OQ101591; CAM: OQ101586-OQ101588; RPB2: OQ101592-OQ101594). A maximum Likelihood (ML) phylogenetic tree was constructed with RAxML version 8.2.10 based on the concatenated sequences (ITS, CAM, TEF1, RPB2). According to morphology and phylogenetic analysis, the isolates were identified as Fusarium sulawesiense (Maryani et al. 2019). For pathogenicity tests, multiple punctures in a 5-mm-diameter circle were made with a sterilized toothpick on detached young healthy fruits, followed by inoculation with 10 μl of conidial suspension (106 spores/ml in 0.1% sterile Tween 20). Each isolate was inoculated onto eighteen fruits. Controls were inoculated with water containing 0.1% sterile Tween 20 under the same conditions. Symptoms were observed on the inoculated fruits 7 days after incubation at 25°C, whereas non-inoculated controls were asymptomatic. The fungus was re-isolated from inoculated chilli fruits, completing Koch's postulates. To our knowledge, this is the first report of Fusarium sulawesiense causing fruit rot on Chilli in China. These results will provide valuable information for prevention and management of fruit rot on Chilli.

Isolation and characterization of halotolerant plant growth promoting rhizobacteria from mangrove region of Sundarbans, India for enhanced crop productivity.

Halotolerant plant growth promoting rhizobacteria (PGPR) are beneficial microorganisms utilized to mitigate the biotic and abiotic stresses in plants. The areas of Sundarban mangroves of West Bengal, India have been reported to be rich in halotolerant microflora, yet major area remains unexplored. The present study, therefore, aims to map down the region-specific native microbial community potent of salt tolerance, plant growth promoting (PGP) activity and antagonistic activity against fungal pathogens. Bacterial samples were isolated from the saline soil of the Sundarban mangroves. A total of 156 bacterial samples were isolated and 20 were screened for their salt tolerance potential. These isolates were characterised using morphological, biochemical, and molecular approaches. Based on 16s rRNA sequencing, they were classified into 4 different genera, including Arthrobacter sp. (01 isolate), Pseudomonas plecoglossicida (01 isolate), Kocuria rosea (01 isolate), and Bacillus (17 isolates). The halotolerant isolates which possessed plant growth promoting traits including phosphate, and zinc solubilization, indole acetic acid production, siderophore, and ammonia generation were selected. Further, the effect of two halotolerant isolates GN-5 and JR-12 which showed most prominent PGP activities was evaluated in pea plant under high salinity conditions. The isolates improved survival by promoting germination (36 to 43%) and root-shoot growth and weight of pea plant in comparison to non-inoculated control plants. In a subsequent dual culture confrontation experiment, both these halo-tolerant isolates showed antagonistic activities against the aggressive root rot disease-causing Macrophomina phaseolina (Tassi) Goid NAIMCC-F-02902. The identified isolates could be used as potential bioagents for saline soils, with potential antagonistic effect on root rot disease. However, further studies at the physiological and molecular level would help to delineate a detail mechanistic understanding of broad-spectrum defence against salinity and potential biotic pathogen.

DETERMINING THE EFFICACY OF DISCLOSURE DYES TO DEMONSTRATE THE PRESENCE OF BIOFILM ON IMPLANT MATERIAL USING BOTH VISUAL AND FLUORESCENT TECHNIQUES IN VITRO

Prosthetic joint infection (PJI) is an important cause of arthroplasty failure. There is no method to disclose the presence or map the distribution of the in vivo biofilm on infected arthroplasty despite the recognition that such a tool would aid intraoperative decision making and improve novel implant design. The aim of this study was to test the efficacy of four dyes to disclose bacterial biofilm in an in vitro setting.Four dyes with known affinity to bacterial biofilm were assessed to determine their efficacy to disclose biofilms in an in vitro model of PJI. Three dyes (Methylene Blue, Indocyanine Green and Rose Bengal) have established clinical utility and the other, Thioflavin T, is known to fluoresce in the presence of amyloid a known biofilm constituent. The efficacy of the dyes to discriminate between biofilms of different mass and vitality (high, low or the non-inoculated control) was determined after three minutes exposure of the biofilm to the dyes by calculating the amount of dye bound to the biofilm via sonication and spectrophotometry, quantification of the dye through standardised photographic imaging of the stained biofilm and the calculation of inter-observer agreement. Each experiment was performed in triplicate for each dye and repeated three times.For each of the disclosure dyes assessed there was significant difference demonstrated between the amount of dye bound to the high and low mass biofilms (p<0.05) as well as in the amount of dye quantified in photographic and fluorescent image assessment between biofilms of differing mass (p<0.01). There was excellent agreement between three observers, for each disclosure dye, in determining the biofilm mass of each stained disc (Kappa>0.91).This study demonstrates the efficacy of biofilm disclosure dyes in an in vitro PJI model which could one day be used to disclose and map the clinical biofilm in vivo.

Field inoculation by arbuscular mycorrhizal fungi with contrasting life-history strategies differently affects tomato nutrient uptake and residue decomposition dynamics

PurposePlant microbial biostimulants, such as arbuscular mycorrhizal fungi (AMF), enhance nutrient concentration in fruits, including tomato. However, field studies on tomato AMF inoculation are scarce. AMF species belonging to Gigasporaceae and Glomeraceae families known to vary in life-history strategies may determine differential effects on plant nutrient benefits and residue decomposition. Despite this, the effect of different life-history strategies on nutrient acquisition of tomato fruits has not been investigated yet.MethodsWe studied the effect of inoculation of two tomato varieties with four AMF species belonging to Glomeraceae and Gigasporaceae. Fungal colonization, yield, fruit nutrient concentration, litter decomposition, and bacterial and fungal abundances in soil were assessed in the field under organic agriculture.ResultsOverall Gigasporaceae promoted the concentration of nutrients in tomato fruits compared to Glomeraceae. A variability in AM fungal colonization and fruit nutrient concentration was detected within Glomeraceae. Scutellospora pellucida increased the yield (+ 27%) of var. Rio Grande with respect to Gigaspora gigantea. In var. Rio Grande, inoculation with Funneliformis mosseae did not change litter decomposition as compared to non-inoculated controls, whereas it was lower than in Sclerocystis sinuosa and Gigasporaceae species, which showed the highest decomposition rates. AMF inoculation promoted soil total bacterial and fungal abundance and fungal:bacterial (F:B) ratio compared to controls, and members of Gigasporaceae had the highest F:B ratio.ConclusionThese findings pointed at the inclusion of AM fungal life-history strategy within the selection criteria for the development of biofertilizers able to enhance the nutritional value of vegetables under organic farming systems.

First Report of Neoscytalidium dimidiatum Causing Stem Canker of Dragon fruit (Hylocereus spp.) in India.

Dragon fruit (Hylocereus spp.) is a tropical perennial plant (Family Cactaceae) that is popular for consumption in India. Originated from Central and South America and currently became popular in India. Hylocereus undatus and H. polyrhizus are the most commonly cultivated species in India. During August to December, 2021, stem cankers were observed in commercial orchards within the Satara, Pune and Solapur districts of Maharashtra. The disease incidence across four orchards was approx. 40% and severity ranged between 30 and 80%. Initial symptoms on infected cladodes were minute, circular, depressed chlorotic spots often with a brick red flecks followed by elevation of the centre of the lesion. Later the lesions turned necrotic and contained black, erumpent pycnidia, followed by chlorosis and stem rot. Twelve diseased cladodes from different orchards were collected and isolations were conducted. Edges of the lesions (5 to10 mm2) were excised and surface sterilized by exposure to 1% sodium hypochlorite (2 min) followed by triple rinsing with sterilized distilled water. Excess moisture was removed with sterilized blotter paper and pieces were plated on potato dextrose agar (PDA) amended with streptomycin sulphate (30 mg/L) for 3 days at 27 ± 2°C with a 12 h photoperiod. All twelve isolation attempts yielded uniform fungal colonies and out of these, purified colonies from each location viz., SLNeo, LNeo, MGNeo and KNeo were selected for etiology study. On PDA, initially white mycelial colonies turned to olive green to grayish with dark gray to black pigmentation. Colony growth was rapid (30 mm/day). Brown, branched septate hyphae fragmented to produce abundant arthroconidia in chains that were hyaline to dark brown, thick walled, 0 to 1 septate, ellipsoid to ovoid (10.1 ± 1.4 × 4.88 ± 1.1 μm), rod (9.0 ± 0.8 μm × 5.3 ± 0.8 μm), round (7.9 ± 1.6 μm) and capsule (14.2 ± 3.1 × 6.1 ± 0.8 μm) shape. Conidia from pycnidia developed on host tissues were aseptate, hyaline and ellipsoid-cylindrical (8.2 ± 0.8 × 2.2 ± 0.3 μm) in shape. Based on morphological characteristics, the isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Molecular characterization was done by amplifying the partial internal transcribed spacer (ITS) region, and translation elongation factor 1-α and β-tubulin genes of four isolates using ITS1/ITS4, EF1-728F/EF1-986R and BT2A/BT2B primers (White et al. 1990, Glass and Donaldson 1995, Carbone and Kohn 1999). Sequences were deposited in GenBank accesions (ITS: OM884028, OM884029, OM884030, OM899800; TUB2: OM927962, OM927963, ON099066; TEF1-α: OM927965, OM927966, OM927964, OM984744) showed 99 to 100% identity with the epitype CBS 499.66 accession numbers KF531820 for ITS and KF531798 for TEF1-α and KF531800 for TUB2. N. dimidiatum isolate designated as SLNeo has been deposited at Microbial Type Culture Collection (MTCC), Chandigarh, India with MTCC 13250. Phylogenetic analysis using maximum likelihood (ML) revealed these isolates were clustered with N. dimidiatum clade. To evaluate pathogenicity, stems of one year old dragon fruit plants were wounded with a sterilized needle and inoculated with 7 mm fresh mycelial discs. For each isolate three replications were kept. Non-inoculated controls were plants receiving only non-colonized PDA discs. Inoculated and control plants were kept separately at ambient temperature (35 ± 2°C during day and 22 ± 2°C at night). Symptom started to develop at 2 days after inoculation (DAI) and canker lesions with chlorotic halos and rotting were observed at 15 DAI. No symptoms were observed on the negative control. Pathogenicity testing was repeated twice and the pathogen was re-isolated from symptomatic tissues with 100% re-isolation frequency and found identical with N. dimidiatum. N. dimidiatum has been reported on H. undatus and H. polyrhizus in subtropical and tropical countries worldwide (Chuang et al. 2012; Ezra et al. 2013; Lan et al. 2012; Sanahuja et al. 2016; Serrato-Diaz and Goenaga 2021). To our knowledge, this is the first report of dragon fruit stem canker caused by N. dimidiatum from India. Since the disease poses a major threat to dragon fruit plantations, additional epidemiological studies may assist in developing management strategies.

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

First Report of Dieback of Quercus suber Trees Associated with Phytophthora quercina in Morocco.

Cork oak (Quercus suber L.) is an evergreen tree native to SW Europe and NW Africa. It covers 2·106 ha in the western Mediterranean basin, forms heterogeneous forest ecosystems and represents an important source of income derived from cork production. While in Iberia, Italy, Tunisia and Algeria, drought and several endemic pathogens have been associated with cork oak decline (Moricca et al. 2016; Smahi et al. 2017), in Morocco there is no evidence, apart from overgrazing and human intervention (Fennane and Rejdali 2015), of a pathogen associated with oak decline. In December 2019, extensive dieback and mortality of 60-year-old cork oak trees were observed in a natural stand of ca 150 ha located 5 km east from Touazithe, in Maâmora forest, Morocco (34°13'38''N, 6°14'51''W - 87 m a.s.l.). Two years before, Q. suber seedlings from a local nursery were planted to increase tree density. Symptoms in trees and planted seedlings included chlorosis, reddish-brown discoloration of the whole crown and dieback starting in the upper crown. Root rot and lack of fine roots were observed. Tree mortality was estimated at ca 30%, and disease incidences of trees and seedlings were 45 and 70%, respectively. A Phytophthora species was consistently isolated from the rhizosphere of 3 symptomatic trees randomly selected at the site using leaves as bait (Jung et al. 1996). On carrot agar Phytophthora colonies were uniform and cottonwool-like. Sporangia were typically terminal, with ovoid, and obpyriform shape, mostly papillate, measuring 30.7 ± 4.7 µm length and 22.7 ± 4.1 µm wide. Oogonia were produced in single culture, and they were globose to subglobose, elongated to ellipsoid, 32.1 ± 2.9 µm in diameter and 46.1 ± 4.8 µm in length. Oospores were usually spherical, thick-walled, and measured 28.1 ± 2.4 µm. Antheridia were paragynous, mostly spherical, measuring 12.2 ± 1.4 µm. Isolates had minimum and maximum temperatures of 5 °C and 30 °C, respectively, and a growth optimum at 20 °C. Apart from the small size of sporangia, features were typical of Phytophthora quercina Jung. The identity of a representative strain (TJ1500) was corroborated by sequencing the ITS and mitochondrial cox1 gene regions, and BLAST search in GenBank showed 100% homology with sequences of the ex-type culture of P. quercina (KF358229 and KF358241 accessions, respectively). Both sequences of the representative isolate were submitted to GenBank (accessions OP086243 and OP290549). The strain TJ1500 is currently stored within the culture collections of the Mendel University in Brno and the University of Sassari. Its pathogenicity was verified and compared with a P. cinnamomi strain in a soil infestation test with one-year-old cork oak seedlings (Corcobado et al. 2017). Five months after inoculation, the symptoms described were observed in the seedlings, and fine root weight of plants inoculated with the TJ1500 strain and P. cinnamomi was reduced by 19 and 42%, respectively, in relation to non-inoculated controls. The pathogen was re-isolated from the necrotic roots, thus fulfilling Koch's postulates. So far, P. quercina has been reported associated with chronic mortality of cork oak in new plantations in Spain (Martín-García et al. 2015; Jung et al. 2016) and natural forests in Italy (Seddaiu et al. 2020). To our knowledge this is the first report of P. quercina in Morocco. Givenat Morocco is an important cork producing country, our finding warns about the risk this pathogen poses to Q. suber and other North African oaks.

First Report of Leaf Spot and Blight on Crown Daisy Caused by Cercospora apii in China.

Crown daisy (Glebionis coronaria L.), also known as chrysanthemum greens, is a popular vegetable in Asia, especially in China. The leaves have been used in folk medicine as a tonic for the liver, blood, intestines and to control anemia and high blood pressure. In November 2020, severe leaf spot and blight was observed with 80% to 95% incidence on crown daisy growing in greenhouses in Fengxian, Shanghai, China(121°22'E, 30°53'N). Irregular rounded spots appeared with a light gray center and water-soaked margins. Round lesions enlarged and merged with age, followed by the development of a necrotic area resulting in the typical "frog-eye" and causing a continuous deterioration of crown daisy. Diseased leaves were washed in running water for 30 min. Small fragments (5 × 5 mm) taken from the margin of lesions were disinfected with 1% NaClO for 3 min, rinsed three times with sterile water, cultured on potato sucrose agar (PSA) augmented with 50 mg streptomycin/liter at 26 oC,and incubated in the dark. Colonies had identical morphology, and TH11290202 was selected and deposited in the plant pathology lab of Shanghai Academy of Agricultural Sciences. Mycelium was initially cottony and white and became appressed to the medium and dark brown with time. Conidia did not form on any media, including PSA, PDA, V8 agar (V8A), maize leaf carbonate agar (MLPCA), pepper leaf carbonate agar (PLPCA), etc. To confirm the identity of the pathogen, genomic DNA was extracted from TH11290202 with the cetyltrimethylammonium ammonium bromide (CTAB) method from the mycelia. Five loci were PCR amplified, namely, the internal transcribed spacer (ITS), translation elongation factor (TEF), calmodulin (cmdA), histone (H3) and actin (ACT), using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Jaklitsch et al. 2005), CAL-F/CAL-R (O'Donnell et al. 2000), cylh3f/cylh3r (Glass and Donaldson 1995), and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. The resulting sequences were deposited in GenBank (MW819910, MW981277, MW981278, ON798723, and MW981279). Analysis of the ITS, TEF, cmdA, H3 and ACT gene sequences of isolate TH11290202 revealed that it was a member of the genus Cercospora, sharing 99.79%, 99.66%, 98.10% 99.74% and 100% sequence similarity with type strain of Cercospora apii CBS 116455. A multilocus phylogenetic analysis was performed using sequences from other closely related taxa obtained from GenBank. Based on morphological and molecular characteristics, TH11290202 was identified as C. apii (Crous and Braun 2003; Groenewald et al. 2006; Milosavljević et al. 2014). To confirm pathogenicity, Koch's postulates were fulfilled on 30 mature plants, which were maintained in a growth chamber (at 26 °C, relative humidity 90%, 12/12 h light/dark). Surface-sterilized leaves were sprayed with a mycelial suspension. Brown lesions were formed 7 days after inoculation on 15 plants, whereas the noninoculated controls remained asymptomatic on the other 15 plants. To our knowledge, this is the first report of C. apii causing leaf spot and blight on G. coronaria in China and will provide useful information for developing effective control strategies.

Matured compost amendment improves compost nutrient content by changing the bacterial community during the composting of Chinese herb residues.

Composting is a sustainable strategy to deal with organic waste. Our research aimed to study the influence of an amendment of 10% matured compost (MC) during Chinese herb residue (CHR) compost. Here, a 60-day CHR compost was performed, and MC application was able to reduce the nitrogen loss and enhance the humic acid accumulation during the composting as compared with the non-inoculated control (NC), by 25 and 19%, respectively. Furthermore, the matured compost amendment improved the diversity of the bacterial community, increased the complexity of the co-occurrence network, and changed the keystone and module hub bacteria during composting. The increased abundance levels of Thermopolyspora, Thermobispora, and Thermosporomyces, which were significantly higher in MC than in NC, may contribute to the degradation of cellulose and the formation of humic acid. Overall, this study extends our understanding of the effects of matured compost reflux on compost quality and the bacterial community.

Fermentation Properties and Bacterial Community Composition of Mixed Silage of Mulberry Leaves and Smooth Bromegrass with and without Lactobacillus plantarum Inoculation

To evaluate the fermentation properties and bacterial community composition of mulberry leaves when ensiled with smooth bromegrass, and the effects of Lactobacillus plantarum inoculation on the mixed silage of mulberry leaves and smooth bromegrass, mulberry leaves were mixed with smooth bromegrass at ratios of 100:0, 90:10, 80:20, 70:30 and 60:40, and ensiled for 60 d with and without L. plantarum inoculant. The results showed that the sole fermentation of mulberry leaves failed to achieve optimum fermentation quality. Silage with a mulberry leaf ratio of 80% performed better fermentation quality compared with other non-inoculated groups, indicated by lower pH value, adequate lactic acid accumulation, and enriched proportion of Lactobacillus in the bacterial community. L. plantarum inoculation dramatically improved fermentation quality of mulberry leaf silage compared with the non-inoculated control. However, the fermentation quality of the inoculated silage decreased along with the reduction in the ratio of mulberry leaves. In conclusion, L. plantarum inoculation has the capability to improve the silage quality of mulberry leaves. Combined ensiling with smooth bromegrass could also aid in improving silage quality of mulberry leaves, with the optimum ratio of mulberry leaves being 80%.

Susceptibility of Ovine Bone Marrow-Derived Mesenchymal Stem Cell Spheroids to Scrapie Prion Infection

In neurodegenerative diseases, including prion diseases, cellular in vitro models appear as fundamental tools for the study of pathogenic mechanisms and potential therapeutic compounds. Two-dimensional (2D) monolayer cell culture systems are the most used cell-based assays, but these platforms are not able to reproduce the microenvironment of in vivo cells. This limitation can be surpassed using three-dimensional (3D) culture systems such as spheroids that more effectively mimic in vivo cell interactions. Herein, we evaluated the effect of scrapie prion infection in monolayer-cultured ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) and oBM-MSC-derived spheroids in growth and neurogenic conditions, analyzing their cell viability and their ability to maintain prion infection. An MTT assay was performed in oBM-MSCs and spheroids subjected to three conditions: inoculated with brain homogenate from scrapie-infected sheep, inoculated with brain homogenate from healthy sheep, and non-inoculated controls. The 3D conditions improved the cell viability in most cases, although in scrapie-infected spheroids in growth conditions, a decrease in cell viability was observed. The levels of pathological prion protein (PrPSc) in scrapie-infected oBM-MSCs and spheroids were measured by ELISA. In neurogenic conditions, monolayer cells and spheroids maintained the levels of PrPSc over time. In growth conditions, however, oBM-MSCs showed decreasing levels of PrPSc throughout time, whereas spheroids were able to maintain stable PrPSc levels. The presence of PrPSc in spheroids was also confirmed by immunocytochemistry. Altogether, these results show that a 3D culture microenvironment improves the permissiveness of oBM-MSCs to scrapie infection in growth conditions and maintains the infection ability in neurogenic conditions, making this model of potential use for prion studies.

Cadmium-absorptive Bacillus vietnamensis 151–6 reduces the grain cadmium accumulation in rice (Oryza sativa L.): Potential for cadmium bioremediation

Microbial bioremediation of heavy metal-polluted soil is a promising technique for reducing heavy metal accumulation in crops. In a previous study, we isolated Bacillus vietnamensis strain 151–6 with a high cadmium (Cd) accumulation ability and low Cd resistance. However, the key gene responsible for the Cd absorption and bioremediation potential of this strain remains unclear. In this study, genes related to Cd absorption in B. vietnamensis 151–6 were overexpressed. A thiol-disulfide oxidoreductase gene (orf4108) and a cytochrome C biogenesis protein gene (orf4109) were found to play major roles in Cd absorption. In addition, the plant growth-promoting (PGP) traits of the strain were detected, which enabled phosphorus and potassium solubilization and indole-3-acetic acid (IAA) production. Bacillus vietnamensis 151–6 was used for the bioremediation of Cd-polluted paddy soil, and its effects on growth and Cd accumulation in rice were explored. The strain increased the panicle number (114.82%) and decreased the Cd content in rice rachises (23.87%) and grains (52.05%) under Cd stress, compared with non-inoculated rice in pot experiments. For field trials, compared with the non-inoculated control, the Cd content of grains inoculated with B. vietnamensis 151–6 was effectively decreased in two cultivars (low Cd-accumulating cultivar: 24.77%; high Cd-accumulating cultivar: 48.85%) of late rice. Bacillus vietnamensis 151–6 encoded key genes that confer the ability to bind Cd and reduce Cd stress in rice. Thus, B. vietnamensis 151–6 exhibits great application potential for Cd bioremediation.

Endophytic fungi improve management of the burrowing nematode in banana (Musa spp.) through enhanced expression of defence-related genes

Summary The root-burrowing nematode, Radopholus similis, is reputedly the most damaging nematode pest of banana and responsible for major production losses. In this study, the endophytic potential of 13 fungal isolates was assessed for the management of R. similis in East African Highland bananas (‘Ng’ombe’). All isolates successfully colonised tissue-cultured banana roots, with isolates from Trichoderma, Fusarium and Hypocrea producing the highest (⩾49.1%) and Beauveria isolates the lowest (⩽14.4%) colonisation. The fungal endophytes T. asperellum (ICIPE 700) and H. lixii (ICIPE 697) were the most effective in reducing R. similis densities (>81%) relative to the non-inoculated control. However, the combined inoculation of ICIPE 700 and ICIPE 697 led to greater suppression of R. similis (>21%) relative to individual inoculation. Suppression of R. similis following inoculation of banana roots with ICIPE 700 and/or ICIPE 697 was associated with the significant upregulation of the defence-related gene PR-1, the cell signalling gene calmodulin Ca2+ and the cell-wall-strengthening gene β-1,3-glucan synthase. This study demonstrates the potential for nematode management in bananas with fungal endophytes, especially using the isolates ICIPE 700 and ICIPE 697 when combined.

First report of the root-knot nematode Meloidogyne incognita on Salvia miltiorrhiza Bunge in Henan Province, China.

First report of the root-knot nematode Meloidogyne incognita on Salvia miltiorrhiza Bunge in Henan Province, China.

Effects of a Microbial Restoration Substrate on Plant Growth and Rhizosphere Microbial Community in a Continuous Cropping Poplar

In poplar cultivation, continuous cropping obstacles affect wood yield and soil-borne diseases, primarily due to structural changes in microbes and fungus infection. The bacterium Bacillus cereus BJS-1-3 has strong antagonistic properties against pathogens that were isolated from the rhizosphere soil of poplars. Poplar rhizospheres were investigated for the effects of Bacillus cereus BJS-1-3 on microbial communities. Three successive generations of soil were used to replant poplar seedlings. BJS-1-3 inoculated poplars were larger, had higher plant height and breast height diameter, and had a greater number of total and culturable bacteria than non-inoculated controls. B. cereus BJS-1-3 inoculated poplar rhizospheres were sequenced, utilizing the Illumina MiSeq platform to analyze changes in diversity and structure. The fungi abundance and diversity in the BJS-1-3 rhizosphere were significantly lower than in the control rhizosphere. In comparison to the control group, Bacillus sp. constituted 2.87% and 2.38% of the total bacterial community, while Rhizoctonia sp. constituted 2.06% and 6.00% of the total fungal community. Among the potential benefits of B. cereus BJS-1-3 in poplar cultivation is that it enhances rhizosphere microbial community structure and facilitates the growth of trees.

Occurrence of root rot caused by Fusarium fujikuroi and Fusarium proliferatum on peanut in China.

Root rot of peanut, caused by Fusarium spp., is a devastating disease in most peanut cultivation regions. In this reported outbreak, Fusarium root rot of peanut has been observed in Henan province, China in July 2021. About 20% of peanuts in a field (0.66 ha) were affected. Early symptoms comprised the wilt of the lower leaves, and the darkening of the vascular tissue of roots, which turned brown. Progressively, the whole plant wilted, the roots rotted, and the plant ultimately died. Pathogenic species were isolated from plants showing symptoms of root rot in the field. A total of 206 Fusarium isolates were generated, and 16 isolates were preliminarily identified as Fusarium fujikuroi based on morphological characteristics. Isolates were obtained and grown on PDA plates. Isolates developed floccose white aerial mycelia with reddish-pink coloration in the medium in 2 weeks on the benchtop. Macroconidia were 3-5 septate, measuring 27.5 to 48.8 × 2.6 to 3.8 μm (avg. 36.7 × 3.6 μm, n=50). Microconidia were abundant in chains, mainly asepatate, oval to kidney-shaped, 4.0 to 11.6 × 2.5 to 4.1 μm (avg. 5.8 ×3.1 μm, n=50). DNA was extracted from mycelium and the following genes were amplified and sequenced: the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al.1990) (Genbank assessions MZ831304 to MZ831308), the partial calmodulin gene (CAM, primer CL1/CL2A, O'Donnell.)(Genbank assession MZ856333 to MZ856337) and the partial translation elongation factor (EF-1α) using primer EF1/EF2 (Geiser et al.)(Genbank assession MZ856338 to MZ8564342). FUSARIUM-ID analysis showed 98.18% to 100% similarity with sequences of the F. fujikuroi species complex. The phylogenetic analysis was conducted using a neighbor-joining algorithm based on the ITS, CAM, and EF-1α gene sequences. The isolates were clustered with F. fujikuroi clade (Supplementary Fig.1). Koch's postulates were conducted using a sand-cornmeal-inoculum-layer method (Bilgi et al.). Briefly, 400 ml plastic boxes were filled with 15g of sterilized premium-grade coarse vermiculite, followed by a 15 g of inoculum prepared as sand-cornmeal mixture inoculum. The inoculum for each F. fujikuroi isolates was prepared by infesting a pre-sterilized sand-cornmeal mixture with three 5 mm plugs of cultures. Three F. fujikuroi isolates and PDA plugs were inoculated to serve as positive control and non-inoculated control treatments, respectively. The completed colonization of the sand-cornmeal mixture was finished by incubating at 25 ℃ for 7-10 days. Eight pre-germinated seeds of cv. Luhua No.1 was then covered with another 8 g of vermiculite. Peanuts were grown at 25 °C with 85% relative humidity under a light/dark cycle of 14h/10h. After 14 days of incubation, the inoculated plants showed typical symptoms of root rot similar to those in the field: pre-emergence damping-off, reddish-brown lesions on the tap, and lateral roots. F. fujikuroi was successfully re-isolated from inoculated plants but not from the controls and identified as described above. F. fujikuroi was reported to cause bakanae disease of rice (Amatulli et. al.), and root rot of soybeans (Zhao et. al.). To the best of our knowledge, this is the first record of F. fujikuroi causing root rot of peanut in China.

A Study of the Effect of Phosphorous Solubilization by Microorganisms on Growing Plants Maize (Zea mays) in Adamawa, Cameroon

Aims: Aims of this study was to improve the growth of Maize (Zea mays) in the Adamawa region specially characterized by Sudano-Guinean and Sudano-Sahelean Savannah area.
 Localization of Site Study: Study was conducted out on Jun 2021 in Bini-Dang, in order to identify suitable strains of Microorganisms Solubilizing Phosphorus (MSP) of roots and rhizosphere of Zea mays. Ten strains of Microorganisms Solubilizing Phosphorus (MSP) of roots and rhizosphere of Zea mays were sampled.
 Methodology: The experimental design was three blocks completely randomized with 132 plots. A total of 11 treatments were considered during the experiment. Different treatments are constituted: BN, NBB, BNB17, RBNBB5, SSL9, SDB6, RKB1, BG12, SDL4, RDB1 and Control) repeated 12 times. Parameters were: germination time, number of leaves, height of plants, diameter of stem, date of flowering and the number of cobs per plant.
 Results: Our results showed that Bacteria solubilizing phosphorous was significantly (P<0.05) improve yields of plants compared to non-inoculated control in all treatments. Germination time, the number of leaves, the height of plants, the diameter of stem, the flowering and the number of cobs per plant were statistically (P<0.05) most important with uses of strains microorganisms solubilizing phosphorous in block 2, which received Natural Phosphorus (NP) and inoculated by Microorganisms Solubilizing Phosphorous in the block 3 (inoculated with MSP).
 Conclusion: Strains of MSP improves the germination, growing and number of cobs per plants of Zea mays in solubilizing Natural Phosphorous (NP). The strains of MSP, BNB7, BNB17, RBNB5, BN and SDB6 were significantly most performants, compared to others strains effect on field. The MSP could be used as biological fertilizers by direct application on soils with Natural Phosphorous (NP).

Reaction of Winter Cover Crops to Meloidogyne enterolobii and Glasshouse Bioassay for Evaluating Utility in Managing M. enterolobii in Soybeans.

Meloidogyne enterolobii is an invasive and highly aggressive root-knot nematode pathogen impacting the Southeastern United States. Winter cover cropping may be a cost-effective method for reducing populations of M. enterolobii in between summer cash crops, yet a gap in the knowledge remains about the response of these cover crops to M. enterolobii and their utility in suppressing nematode populations prior to a cash crop. A "two-step" glasshouse bioassay was performed to evaluate eight winter cover crops popular in North Carolina for their direct response to M. enterolobii infection, and to quantify their effect in reducing nematode populations for the following soybean plants. Data on cover crop root galling, soybean root galling, soybean shoot fresh weight, soybean root fresh weight, eggs per gram of soybean root, and a modified reproductive factor were collected. Cereal cover crops did not display root galling, and there was significantly less root galling in those soybean plants following cereal winter cover crops when compared to those following broadleaf winter cover crops. Broadleaf winter cover crops resulted in significantly higher eggs per gram of soybean root and modified reproductive factor in the soybean plants, compared to cereal cover crops and non-inoculated controls. Results from this study suggest that cereal winter cover crops may be poor-hosts to M. enterolobii and may significantly reduce M. enterolobii populations before a soybean crop, compared to broadleaf winter cover crops. This study lays the groundwork for management recommendations and future field trials to assess management of M. enterolobii through winter cover cropping.

First Report of Fusarium ipomoeae causing Fusarium wilt on Glycine max in South Korea.

Soybean (Glycine max L.) is one of the most important crops worldwide. In South Korea, three species of Fusarium have been reported as causal pathogens of Fusarium wilt of soybean (KSPP, 2021). From 2017 to 2018, wilted soybeans were observed in two soybean fields in Daegu (36.62°126.91°) and Yesan (35.89°128.44°), South Korea. The incidence rate was about 2 to 5% of the total 0.1ha, respectively. The diseased soybeans were yellowed from the lower leaves or dried up, and the inside of the root and stem were turned brown. Fragments (each 5 mm × 5 mm) of the symptomatic vascular tissue were surface-sterilized with 1% NaOCl for 1 min, and then rinsed twice in sterilized distilled water. The seven pieces each from two diseased plants were placed on water agar and incubated at 25°C for 5 days. Two single spore isolates were cultured on carnation leaf agar at 25°C for 14 days under near ultra violet/dark conditions for 12 hours. Macroconidia of two isolates were mostly 3- to 5-septate, dorsiventral curvature, hyaline, apical cell hooked to tapering, basal cell foot-shaped, and measured 51.3 - 62.2 × 3.7 - 4.7 μm (DG43821) and 63.8-74.8×3.1-4.4 μm (YS37232). Microconidia were not observed. Chlamydospores were produced in chains or pairs, subglobose and thick walled. The color of the aerial mycelium was pinkish white and the reverse of the colony was brownish orange on potato dextrose agar. Based on morphological and cultural characteristics, the two isolates were identified as belonging to Fusarium incarnatum-equiseti species complex (Leslie and Summerell 2006). To confirm the accurate species identification of the two isolates, DNA sequencing of the internal transcribed spacers and intervening 5.8S (ITS), partial translation elongation factor 1-alpha (TEF) and RNA polymerase II largest subunit (RPB2) genes was carried out using primer sets of ITS1/ITS4, EF1 / EF2 and 7cf / 11ar, respectively (O'Donnell et al. 2010). The nucleotide sequences obtained of two isolates were deposited in GenBank with accession numbers of MW375694, MW375695, MW382963, MW382964, MZ364324 and MZ364325. Identities of the ITS region, TEF and RPB2 gene sequences of the two isolates were 490/492, 482/483, 632/633, 631/632, 870/870 and 931/931 with those of ex-type strain F. ipomoeae LC12165 (MK280832, MK289599 and MK289752) in GenBank, respectively. Thus, based on molecular characteristics, the two isolates were confirmed as F. ipomoeae. A pathogenicity test of the two isolates was conducted using root-dip inoculation on seedlings of one soybean cultivars, Pyeongwon. A spore suspension was prepared by flooding 10-day-old cultures on PDA with sterilized distilled water. Fifteen soybean seedlings at the VC stage per each isolate were inoculated by dipping the roots in the spore suspension (1 × 106 conidia/mL) for 2 hours. Inoculated plants were transplanted into pots containing sterilized soil and maintained in the greenhouse at 28±3°C with 14 h/10 h light/dark. An equal number of plants inoculated with sterilized distilled water served as controls. Five days after inoculation, withered symptoms were observed on two or four of the inoculated seedlings, and by 10 days after inoculation, all inoculated plants had withered and died. No symptoms were observed in the non-inoculated control soybeans. The pathogen was consistently re-isolated from only inoculated plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing Fusarium wilt on soybean in South Korea, as well as worldwide. This pathogen has been reported on peanut in China as a causal agent of leaf spot (Xu et al., 2021). Understanding the host range of this pathogen and the distribution of F. ipomoeae affecting legume crops in South Korea is important, to ensure an effective management of Fusarium wilt on soybeans.