Virens Isolates Research Articles

Conformation Techniques for False Smut Pathogen (U. virens) on Rice through Cultural and Molecular Tools in Telangana

The study was conducted during period of January to April, 2022 at the Department of Plant Pathology and Institute of Biotechnology, Professor Jayashankar Telangana State Agricultural University, Rajendranagar, Hyderabad, Telangana state, India. The main objective of this study was to investigate cultural and morphological characteristics of U. virens isolates on potato sucrose medium (PSA) and their molecular confirmation collected from rice growing regions of Telangana during the kharif (rainy season), 2021. The isolates showed varying colony diameters and growth rates, with Uv3 reaching a maximum diameter of 77 mm and a growth rate of 2.85 mm, while Uv11 exhibited a minimum diameter of 40 mm with a growth rate of 1.48 mm. Notably, twenty two isolates showed diverse characteristics, including colony color, elevation, zonation, sectoring, and chlamydospore formation. Cluster analysis revealed the formation of five major groups, each group containing isolates with distinct features and PCR amplification with universal and species-specific primers yielded products of 700 bp and 380 bp, respectively. Phylogenetic tree analysis, indicated, Uv3 and Uv15 belonged to clade X and clade VII, respectively, both comprising Indian isolates. In contrast, Uv4 was grouped under clade II, which included both Chinese and Indian isolates. The proposed technique coupled with information of U. virens will immensely contribute to further studies on this fungus and false smut disease in India.

Sensitivity Baselines, Resistance Monitoring, and Molecular Mechanisms of the Rice False Smut Pathogen Ustilaginoidea virens to Prochloraz and Azoxystrobin in Four Regions of Southern China.

Rice false smut caused by Ustilaginoidea virens is one of the most devastating fungal diseases of rice (Oryza sativa) worldwide. Prochloraz and azoxystrobin belong to the groups of demethylation inhibitors and quinone outside inhibitors, respectively, and are commonly used for controlling this disease. In this study, we analyzed the sensitivities of 100 U. virens isolates from Yunnan, Sichuan, Chongqing, and Zhejiang in Southern China to prochloraz and azoxystrobin. The ranges of EC50 for prochloraz and azoxystrobin were 0.004-0.536 and 0.020-0.510 μg/mL, with means and standard errors of 0.062 ± 0.008 and 0.120 ± 0.007 μg/mL, respectively. However, the sensitivity frequency distributions of U. virens to prochloraz and azoxystrobin indicated the emergence of subpopulations with decreased sensitivity. Therefore, the mean EC50 values of 74% and 68% of the isolates at the main peak, 0.031 ± 0.001 and 0.078 ± 0.004 μg/mL, were used as the sensitivity baselines of U. virens to prochloraz and azoxystrobin, respectively. We found significant sensitivity differences to azoxystrobin among different geographical populations and no correlation between the sensitivities of U. virens to prochloraz and azoxystrobin. Among 887 U. virens isolates, the isolate 5-3-1 from Zhejiang showed moderate resistance to prochloraz, with a resistance factor of 22.45, while no nucleotide variation in the 1986-bp upstream or 1827-bp gene regions of CYP51 from 5-3-1 was detected. Overexpression of CYP51 is probably responsible for its resistance to prochloraz. Finally, artificial inoculation showed that 5-3-1 was highly pathogenic to rice, suggesting that the resistance of U. virens to prochloraz must be monitored and managed in Zhejiang.

Genetic Diversity and Pathogenic Variation of the Rice False Smut Pathogen Ustilaginoidea virens from Different Rice Cultivars.

Rice false smut, caused by Ustilaginoidea virens, has become one of the most devastating grain diseases of rice worldwide. Understanding the genetic diversity of U. virens is essential for efficient disease control and breeding for disease resistance. However, little is known about the genetic variation of U. virens from different rice cultivars. We investigated the genetic diversity and pathogenic variation of U. virens isolates from ten rice cultivars in Zhejiang, China. A total of 260 polymorphic loci and 27 haplotypes were identified based on the 2137-bp combined DNA fragments of all individuals; hap_4 was the most common haplotype, represented by 41 isolates. Phylogeny indicated that all isolates were divided into four genetic groups. Group I was the largest, with 98 isolates, distributed mainly in eight cultivar populations, whereas 90% of the isolates collected from a Changxiang cultivar were clustered in Group Ⅳ. Furthermore, the pairwise FST values exhibited significant genetic differentiation in 27 of the pairwise comparisons between populations, accounting for 23.21% of the total genetic variation. The genetic composition of the isolates of the CX population was distinguishable from that of the other nine populations, and genetic recombination was found in a few isolates. Finally, 27 haplotype representative isolates showed high variation in pathogenicity, and the isolates from the genetic subpopulation Ⅰ were likely to be more virulent than those from genetic subpopulations Ⅱ and Ⅲ. Collectively, these findings suggest that differences in rice cultivars play an important role in the genetic variation of U. virens.

Development and application of recombinase polymerase amplification for rapid detection of rice false smut pathogen (Ustilaginoidea virens)

Ustilaginoidea virens (Cke.) Tak. is the causal pathogen of rice false smut, a grain disease which affects both grain quality and yield. In this study, a recombinase polymerase amplification (RPA) assay protocol was developed to detect U. virens directly from rice spikelet. Three pairs of primer were designed from the U. virens GTP binding protein beta subunit (UvG-β1) gene to function both in RPA and in polymerase chain reaction (PCR). The developed RPA assay efficiently detected U. virens from both genomic DNA and mycelial crude extract. A comparison of sensitivity of RPA with PCR and loop-mediated isothermal amplification (LAMP) revealed that the latter two methods failed to detect U. virens from crude extract. The specificity of the RPA primers was evaluated using DNA from other rice pathogens, as well as, by sequencing the RPA amplicons. For validation, the RPA assay was evaluated on 32 different U. virens isolates from eastern and north-eastern India, as well as, on field samples. The developed RPA assay was able to detect U. virens directly from crude extract prepared from infected rice spikelet. The developed RPA assay will be effective for field monitoring of U. virens.

Prevalence of Carbendazin Resistance in Field Populations of the Rice False Smut Pathogen Ustilaginoidea virens from Jiangsu, China, Molecular Mechanisms, and Fitness Stability.

Rice false smut (RFS), caused by Ustilaginoidea virens, is an important fungal disease of rice. In China, Methyl Benzimidazole Carbamates (MBCs), including carbendazim, are common fungicides used to control RFS and other rice diseases. In this study, resistance of U. virens to carbendazim was monitored for three consecutive years during 2018 to 2020. A total of 321 U. virens isolates collected from Jiangsu Province of China were tested for their sensitivity to carbendazim on PSA. The concentration at which mycelial growth is inhibited by 50% (EC50) of the carbendazim-sensitive isolates was 0.11 to 1.38 µg/mL, with a mean EC50 value of 0.66 μg/mL. High level of resistance to carbendazim was detected in 14 out of 321 isolates. The resistance was stable but associated with a fitness penalty. There was a statistically significant and moderate negative correlation (r= -0.74, p < 0.001) in sensitivity between carbendazim and diethofencarb. Analysis of the U. virens genome revealed two potential MBC targets, Uvβ1Tub and Uvβ2Tub, that putatively encode β-tubulin gene. The two β-tubulin genes in U. virens share 78% amino acid sequence identity, but their function in MBC sensitivity has been unclear. Both genes were identified and sequenced from U. virens sensitive and resistant isolates. It is known that mutations in the β2-tubulin gene have been shown to confer resistance to carbendazim in other fungi. However, no mutation was found in the Uvβ2Tub gene in either resistant or sensitive isolates. Variations including point mutations, non-sense mutations, codon mutations, and frameshift mutations were found in the Uvβ1Tub gene from the 14 carbendazim-resistant isolates, which have not been reported in other fungi before. Thus, these results indicated that variations of Uvβ1Tub result in the resistance to carbendazim in field isolates of Ustilaginoidea virens.

Cultural Characteristics and Molecular Confirmation of Ustilaginoidea virens the Incitant of False Smut of Rice

Aims: Ustilaginoidea virens, which causes false smut of rice (synonyms: green smut or pseudo smut), is an emerging grain disease around the world. The main aim of the study is to find out suitable culturable medium, carbon sources and pH for the growth of false smut pathogen and molecular confirmation of U. virens isolates.
 Place and Duration of Study: The in vitro studies were conducted in the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, between December 2020 to April 2021.
 Methodology: To study the growth pattern of U.virens, five different carbon sources and mediums were used. Standardization of pH was done using a PSA medium amended with different pH levels. DNA extraction of all 13 isolates were done using the CTAB method. The phylogenetic tree was constructed using MEGA 12 software.
 Results: It is a slow-growing pathogen and grew well in the potato sucrose agar medium with a mycelial growth of about (54.00 mm) after 30 days and sucrose was found to be a good carbon source. The pH of 5 was found to be optimum. PCR reaction was carried out to further validate 13 isolates using three sets of primer pairs ITS 1/ITS 4, US1-5/US3-3, and US 2-5/US 4-3 with amplified products of 560 bp, 380 bp and 260 bp. Accession numbers were obtained for all the isolates.

The Toxicity of Salicylhydroxamic Acid and Its Effect on the Sensitivity of Ustilaginoidea virens to Azoxystrobin and Pyraclostrobin.

Rice false smut (RFS) caused by Ustilaginoidea virens has been one of the most severe rice diseases. Fungicide-based chemical control is a significant measure to control RFS. In the sensitivity determination of quinone outside inhibitor (QoI) fungicide in vitro, salicylhydroxamic acid (SHAM) has been commonly added to artificial culture media in order to inhibit alternative oxidase of phytopathogenic fungi. However, some studies showed that artificial media should not include SHAM due to its toxicity. Whether SHAM should be added in the assay of U. virens sensitivity to QoI fungicide remains unknown. In this study, two appropriate media, potato sucrose agar (PSA) and minimal medium (MM), were selected to test SHAM toxicity and sensitivity of U. virens to azoxystrobin and pyraclostrobin. The mycelial growth and sensitivity to azoxystrobin and pyraclostrobin had no significant difference between on PSA and MM. SHAM could significantly inhibit mycelial growth, conidial germination, peroxidase (POD) and esterase activity of U. virens. Average effective concentration for inhibiting 50% (EC50) values of SHAM against mycelial growth of ten U. virens were 27.41 and 12.75 μg/mL on PSA and MM, respectively. The EC50 values of SHAM against conidial germination of isolates HWD and JS60 were 70.36 and 44.69 μg/mL, respectively. SHAM at 30 μg/mL significantly inhibited POD and esterase activity of isolates HWD and JS60, and even SHAM at 10 μg/mL significantly inhibited POD activity of isolate HWD. In addition, SHAM significantly reduced EC50 values and EC90 values of azoxystrobin and pyraclostrobin on both PSA and MM. Even in the presence of SHAM at 10 μg/mL, average EC50 values of ten U. virens isolates for azoxystrobin decreased 1.7-fold on PSA and 4.8-fold on MM, and for pyraclostrobin that decreased 2.8-fold on PSA and 4.8-fold on MM. Therefore, these results suggest that SHAM should not be included in artificial media in the assay of U. virens sensitivity to QoI fungicides.

Assessing the molecular variability in Ustilaginoidea virens, the rice false smut pathogen with ISSR markers

Rice false smut disease is gaining importance because of its impact on the grain yield and its toxin production ability. Fifty-eight isolates of Ustilaginoidea virens were collected from different rice-growing regions of India. DNA of U. virens was isolated by the CTAB method and fifty ISSR primers were screened for molecular variability studies. Twelve primers viz., UBC series 807, 808, 809, 810, 811, 812, 834, 835, 836, 840, 841, and 842 were selected to study the genetic variability. Different parameters of tested primers viz., heterozygosity (Hn), polymorphism information content (PIC), effective multiplex ratio (EMR), marker Index (MI), and resolving power (RP) were calculated. Primers UBC 812 and UBC 809 recorded maximum heterozygosity (Hn). The PIC values ranged from 0.10 to 0.27 and UBC 807 recorded the maximum value of 0.27. The EMR value varied from 6.75 to 24.0, Similarly, UBC 807 recorded the highest value of MI (24) and RP (8.55). A dendrogram was generated using the DARwin software (version 6.0.21A) based on the unweighted neighbor-joining cluster method. All the fifty-eight U. virens isolates were grouped into three major clusters. Clusters I and II had 21 and 35 U. virens isolates respectively. Cluster III had only two isolates. The isolates showed genetic variations and there was no specific grouping based on the geographical distance.

Culture condition and variation in the pathogenicity of Ustilagonoidea virens isolates causing false smut disease of rice

False smut of rice caused by Ustilagonoidea virens was previously considered a minor disease of rice or rather a sign of bumper harvest, hence the disease was also known by the name Lakshmi disease. However, the disease is gaining much importance at present and is reported to occur in most of the rice growing areas of world in moderate to severe forms. Rice which is cultivated throughout the year in West Bengal is also observed to produce the symptoms of the disease every year in varying degree of severity. Effect of light and different temperature on mycelial growth of Ustilagonoidea virens in BOD incubator was tested. It was found that temperature of 28oC and dark condition favors mycelial growth. Twelve different isolates of Ustilagonoidea virens collected from different districts of West Bengal were isolated in laboratory and tested for their variability in Pathogenicity using the susceptible rice variety Swarna (MTU 7029). 2 ml of inoculum suspension of each isolates were injected to rice plant at booting stage with three replications, whereas in control plants 2 ml of sterilized water was injected and percent grain infection was calculated and analyzed. All the inoculated plants were observed to produce typical false disease symptom with varying degree of severity indicating variation in pathogenicity among the isolates. Percent grain infection ranged from 1.461% to 7.936%. Isolate Uv5 from Alipudwar district was found to be significantly more virulent as compared to other isolates with 7.936% grain infection followed by isolate Uv4 (Jalpaiguri district) and Uv3(Nadia district)  with 5.050% and 4.557% grain infection respectively. Significantly lower infection was observed in isolate Uv9 collected from Coochbehar district with 1.461% grain infection followed by isolate Uv2 (North 24 Parganas district) and Uv12 (Hooghly district) with 1.912 and 3.279% grain infection.

Loop-Mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens.

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicles of rice. V. virens is a heterothallic ascomycete controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed that sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect the mating type of V. virens easily and rapidly by using specific primers based on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay used only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that it could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1 and 200.0 pg of MAT1-2) and was 10 times more sensitive than PCR. In addition, we demonstrated the application of mating type via LAMP assay by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggest that it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

Genetic Diversity and Population Structure of the Rice False Smut Pathogen Ustilaginoidea virens in the Sichuan-Chongqing Region.

Rice false smut caused by Ustilaginoidea virens is one of the most devastating fungal diseases of rice panicles worldwide. In this study, two novel molecular markers derived from single nucleotide polymorphism-rich genomic DNA fragments and a previously reported molecular marker were used for analyzing the genetic diversity and population structure of 167 U. virens isolates collected from nine areas in the Sichuan-Chongqing region, China. A total of 62 haplotypes were identified, and a few haplotypes with high frequency were found and distributed in two to three areas, suggesting gene flow among different geographical populations. All isolates were divided into six genetic groups. Groups I and VI were the largest, with 61 and 48 isolates, respectively. The pairwise FST values showed significant genetic differentiation among all compared geographical populations. Analysis of molecular variance showed that intergroup genetic variation accounted for 40.17% of the total genetic variation, while 59.83% of genetic variation came from intragroup genetic variation. The unweighted pair-group method with arithmetic means dendrogram and population structure revealed that the genetic composition of isolates collected from Santai, Nanchong, Yongchuan, and Wansheng dominated by the same genetic subgroup was different from those collected from other areas. In addition, genetic recombination was found in a few isolates. These findings will help to improve the strategies for rice false smut management and resistance breeding, such as evaluating breeding lines with different isolates or haplotypes at different elevations and landforms.

Genetic diversity and population structure analysis of rice false smut pathogen in North India using molecular markers

AbstractFalse smut, caused by Ustilaginoidea virens, is one of the emerging diseases of rice in the world. The fungi show a high degree of morphological and cultural variability and produce diverse symptoms. India is a vast country with 44‐million‐ha rice production area and has only one report on genetic diversity and population analysis of U. virens from the eastern and north‐eastern region. Ten polymorphic RAPD and 21 SSR markers were used to analyse the genetic diversity and population structure of 81 isolates of U. virens from northern India, the second largest rice‐growing region. In this study, higher Polymorphic Information Content (PIC) and Quality Nature of Data (QND) data in simple‐sequence repeats (SSR) markers over random amplified polymorphic DNA (RAPD) revealed its higher efficacy; thus, SSR markers are highly informative and can help to dissect the genetic structures of U. virens. All markers yielded a total of 180 alleles with 1.0 effective alleles per loci. Genetic diversity ranged from 0.1 to 0.39. The principal coordinate analysis (PCoA), and structure analysis identified two genetic clusters of U. virens isolates with some degree of distinctness according to locations. But clusters, PCoA and structure analysis did not group the isolates according to their geographical origin and this may be due to high gene flow (Nm = 1.14).

Changes in Volatile Organic Compounds from Salt-Tolerant Trichoderma and the Biochemical Response and Growth Performance in Saline-Stressed Groundnut

Soil salinity is one of the major obstacles that is limiting the growth and yield of groundnut. This study aims to investigate the effect of growth-promoting fungi, Trichoderma, on groundnut plants that were cultivated in saline conditions. Five different Trichoderma isolates were grown in four different NaCl concentrations. Selected Trichoderma were then applied to the groundnut seeds and their growth and development were monitored during the study. Growth inhibition, volatile organic compounds, chlorophylls, carotenoids, total phenolics and flavonoids, and minerals were assessed between the Trichoderma treatments. Increasing the salt concentration from 0.25–0.75 M decreased the growth of the Trichoderma isolates. The amounts and profiles of the volatile organic compounds from the T. asperellum isolate were significantly different to those in the T. virens isolate. In the vegetative growth stage, increased chlorophyll content was recorded in both the T. asperellum and T. virens-treated groundnut. The leaves that were obtained from the groundnut that was treated with T. virens T.v4 contained significantly higher indole-3-acetic acid (420 µg IAA/g) than the same plants’ roots (113.3 µg IAA/g). Compared to the control groundnut, the T. asperellum T.a8-treated groundnut showed increased phenolics (31%) and flavonoids (43%) and increased shoots and biomass weight at the generative growth stage. This study demonstrates that Trichoderma, with their plant growth promotion ability, could potentially be used to improve the growth of groundnut growing under salinity stress. Importantly, salt-tolerant Trichoderma could be regarded as a beneficial and sustainable way to improve the survival of salt-sensitive crops.

High-Quality Genome Sequence Resource of a Rice False Smut Fungus Ustilaginoidea virens Isolate, UV-FJ-1.

Ustilaginoidea virens is the fungal pathogen causing rice false smut, resulting in not only yield lost but also grain pollution with toxic mycotoxins. Here we deployed PacBio Sequel II HIFI-read sequencing technology to generate a near-complete genome assembly for the U. virens isolate UV-FJ-1 (38.48 Mb), which was isolated from Fujian province, China. The genome assembly contains 116 contigs with N50 of 0.65 Mb and a maximum length of 2.10 Mb, and the genome completeness is ≥98% assessed by benchmarking universal single-copy orthologs (BUSCOs) and the mapping rate of Illumina short reads. Excluding 35.78% repeat sequences, we identified a total of 7,164 protein-coding genes, of which 5,818 were functionally annotated and 223 encode putative effector proteins. Moreover, 21 secondary metabolite biosynthesis gene clusters were found in UV-FJ-1 genome. Taken together, this high-quality genome assembly and gene annotation resource will provide a better insight for characterizing the biological and pathogenic mechanisms of U. virens.

Monitoring and analysis of rice pathogen Ustilaginoidea virens isolates with resistance to sterol demethylation inhibitors in China

Rice false smut (RFS), caused by Ustilaginoidea virens (Cooke) Takah, is an important fungal disease of rice. In China, sterol demethylation inhibitors (DMIs) are common fungicides used to control RFS. In a previous study, we detected two propiconazole-resistant U. virens isolates in 2015 in Huai’an city, Jiangsu Province, China. In the current study, we detected six propiconazole-resistant isolates out of 180 U. virens isolates collected from rice fields in Jiangsu Province in 2017, and found they were from three different places (Xuzhou, Huai’an and Jintan). All these six propiconazole-resistant isolates were cross-resistant to three other sterol demethylation inhibitor (DMI) fungicides, i.e. difenoconazole, tebuconazole, and epoxiconazole. Among them, two isolates (2017–61 and 2017–170) had high fitness. Through sequencing and RT-qPCR analysis, we found that the expression levels of CYP51 and its encoded protein were significantly increased in the propiconazole-resistant isolates with a “CC” insertion mutation upstream of the CYP51 coding region compared to the propiconazole-sensitive isolates. In addition, propiconazole stimulated CYP51 expression in all isolates. Propiconazole also stimulated the accumulation of CYP51 protein in propiconazole-sensitive isolates and propiconazole-resistant isolates without mutation, but not in propiconazole-resistant isolates with the “CC” mutation. According to JASPAR database analysis, the predicated functional binding sites for propiconazole-resistant isolates with a “CC” insertion mutation and propiconazole-sensitive isolates were different. Given the high fitness of the propiconazole-resistant isolates, the development of resistance to DMIs in U. virens should be monitored. Furthermore, we speculated that the over-expression of CYP51 may contribute to DMI resistance in U. virens with the “CC” insertion mutation.

The Conserved Effector UvHrip1 interacts with OsHGW, and Infection of Ustilaginoidea virens Regulates Defense- and Heading Date-Related Signaling Pathway.

Ustilaginoidea virens, which causes rice false smut (RFS), is one of the most detrimental rice fungal diseases and poses a severe threat to rice production and quality. Effectors in U. virens often act as a group of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of individual effectors in U. virens need to be further explored. Here, we demonstrated a small secreted hypersensitive response-inducing protein (hrip), named UvHrip1, which was highly conserved in U. virens isolates. UvHrip1 was also proven to suppress necrosis-like defense symptoms in N. benthamiana induced by the oomycete elicitor INF1. The localization of UvHrip1 was mainly in the nuclei and cytoplasm via monitoring the UvHrip1-GFP fusion protein in rice cells. Furthermore, Y2H and BiFC assay demonstrated that UvHrip1 interacted with OsHGW, which is a critical regulator in heading date and grain weight signaling pathways in rice. Expression patterns of defense- and heading date-related genes, OsPR1#051 and OsMYB21, were down-regulated over U. virens infection in rice. Collectively, our data provide a theory for gaining an insight into the molecular mechanisms underlying the UvHrip1 virulence function.

Diversity analysis of northern Indian isolates of Ustilaginoidea virens

False smut of rice (Ustilaginoidea virens) (Cooke) Takahashi has become an important disease causing significant yield losses in major rice producing states of India. The present study is focused on morphological, pathological and genetic characterization in different isolates of U. virens. Thirty-five isolates of U. virens collected from different areas of Punjab and adjoining states were used to study the variability. All isolates produced well-defined colonies on potato sucrose agar (PSA) medium with colony diameter ranging from 26.0 to 90.0 mm after 2 weeks of incubation. Colony color in most of the isolates was initially white which changed to yellow and finally became green. The conidia were spherical, hyaline and warty with a size ranging from 4.05–4.94 to 6.10–6.36 μm. Isolates also differed in the growth pattern from appressed, fluffy, less fluffy to raise. Among all isolates, Uv1 was found more aggressive on both rice cultivars tested (PR116 and PR114).Three isolates viz. Uv17, Uv26 and Uv28 failed to produce disease on PR 114 while the rest of the isolates differed in their virulence/aggressiveness behaviour on PR 116 and PR 114. The genetic variation among 35 isolates of U. virens by PCR amplification using ten random operon decamer primers showed polymorphism. Similarity coefficient values among U. virens isolates ranged from 0.56 to 0.97.

Biodiversity and Molecular Characterization of Trichoderma spp. and Exploring its Synergistic Action for the Management of Cucumber Damping Off Incited by Pythium aphanidermatum

Bioversity of fungal antagonist, Trichoderma from different locations of Nilgiri district of Tamil Nadu, India, were characterized through molecular methods. Thirty four isolates were tentatively identified as Trichoderma and confirmed upto species level through molecular tools. PCR amplification of the 18s-28s rRNA gene region revealed that all thirty four isolates produced amplicon size of 600bp and were further confirmed through sequencing. The sequences of various Trichoderma spp. were compared with both NCBI and TrichOKEY database to validate their molecular identity. Among 34 isolates, 31 isolates were identified as T. asperellum (KT462693, KU361372, KX533978 to KX533999, KX523262 to KX523264, KX555650, KX147092 to KX147094, KX5334000), 2 isolates as T. harzianum (KX533989, KX533990) and 1 isolate as T. virens (KU666466) through NCBI data base. However, all the T. asperellum isolates identified through NCBI database were identified as T. koningiopsis using TricHOKEY data base. Identity of T. harzianum isolates (TRI 35 and TRI 36) and T. virens isolate (TRI 37) were same in both NCBI and TricHOKEY database. Antagonistic assay with diverse species of Trichoderma revealed that T. virens (TRI 37) was effective in inhibiting the radial growth of Pythium aphanidermatum (87.78%) followed by T. harzianum (TRI 35), (TRI 36) and T. asperellum (TRI 9) in vitro. The effective isolates T. virens (TRI 37), T. harzianum (TRI 35, TRI 36) and T. asperellum (TRI 9) were compatible with each other. Biopriming of cucumber seeds with talc based formulation of the consortia comprising of T. virens isolate ( TRI 37), T. harzianum isolates (TRI 35 and TRI 36) and T. asperellum TRI 9 @ 108 cfu/g and soil application suppressed damping off to an extent of 76.82% over untreated control.

Effect of Paecilomyces lilacinus, Trichoderma harzianum and Trichoderma virens fungal extracts on the hatchability of Ancylostoma eggs

BackgroundAncylostoma species have demanded attention due to their zoonotic potential. The use of anthelmintics is the usual method to prevent environmental contamination by Ancylostoma eggs and larvae. Nematophagous fungi have been widely used in their biological control due to the fungus ability to capture and digest free nematode forms. AimsThe aim of this study was to evaluate the effect of four different fungal extracts of Paecilomyces lilacinus (n=2), Trichoderma harzianum (n=1) and Trichoderma virens (n=1) isolates on the hatchability of Ancylostoma eggs. MethodsFungal extracts consisted of fungal broth culture supernatant without filtration (crude extract) and filtered broth (filtered extract), macerated mycelium (crude macerate), and macerated mycelium submitted to filtration (filtered macerate). The Ancylostoma eggs were obtained from the feces of naturally infected dogs. In vitro assays were performed in five replicates and consisted of four treatments and one control group. ResultsThe activity of the fungal extracts of each evaluated fungus differed (p<0.05) from those of the control group, showing significant ovicidal activity. The hatching of the eggs suffered reduction percentages of 68.43% and 47.05% with P. lilacinus, and 56.43% with T. harzianum, when crude macerate extract was used. The reduction with the macerate extract of T. virens was slightly lower (52.25%) than that for the filtered macerate (53.64%). ConclusionsThe results showed that all extracts were effective in reducing the hatchability of Ancylostoma eggs. The ovicidal effect observed is likely to have been caused by the action of hydrolytic enzymes secreted by the fungi.

Arabidopsis thaliana: A Model Host Plant to Study Plant-Pathogen Interaction Using Rice False Smut Isolates of Ustilaginoidea virens.

Rice false smut fungus which is a biotrophic fungal pathogen causes an important rice disease and brings a severe damage where rice is cultivated. We established a new fungal-plant pathosystem where Ustilaginoidea virens was able to interact compatibly with the model plant Arabidopsis thaliana. Disease symptoms were apparent on the leaves of the plants after 6 days of post inoculation in the form of chlorosis. Cytological studies showed that U. virens caused a heavy infestation inside the cells of the chlorotic tissues. Development and colonization of aerial mycelia in association with floral organ, particularly on anther and stigma of the flowers after 3 weeks of post inoculation was evident which finally caused infection on the developing seeds and pod tissues. The fungus adopts a uniquely biotrophic infection strategy in roots and spreads without causing a loss of host cell viability. We have also demonstrated that U. virens isolates infect Arabidopsis and the plant subsequently activates different defense response mechanisms which are witnessed by the expression of pathogenesis-related genes, PR-1, PR-2, PR-5, PDF1.1, and PDF1.2. The established A. thaliana–U. virens pathosystem will now permit various follow-up molecular genetics and gene expression experiments to be performed to identify the defense signals and responses that restrict fungal hyphae colonization in planta and also provide initial evidence for tissue-adapted fungal infection strategies.