First report of Phytopythium vexans causing gummosis and root rot of Khasi Mandarin (Citrus reticulata Blanco) in north eastern states of India.

Khasi mandarin (Citrus reticulata Blanco) is the most economically important crop among the citrus growing region in the north-eastern India (Singh et al. 2016). An extensive survey was conducted to identify the causal agent of citrus root rot and gummosis in north eastern states (Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam) of India during October 2021-23. The gummosis disease incidence ranged from 5 to 95 % in 10 to 25 years old Khasi mandarin plants showing relatively more chronic symptoms on mature trees. Yellowing and dropping of leaves, twigs die back, gum oozing from infected bark and loss of feeder roots were the typical symptoms of the disease. Infected bark tissue and young lemon leaf baits in rhizosphere soil were plated on corn meal agar medium supplemented with pimaricin (10 µg/ml), ampicillin (250 µg/ml), rifamycin (10 µg/ml) and 300µg/ml carbendazim and incubated at 26℃. Fifty isolates were purified and maintained on Carrot agar medium. These isolates showed similar cultural and morphological characteristics. Two representative isolates from Arunachal Pradesh (AP21 and AP26) were selected for further experiments and deposited to Indian Type culture collection (ITCC), New Delhi with accession no. 9156 and 9157 respectively. The colonies were fast growing, showing rosette pattern along with whitish blooming mycelium appearance with no visual sporulation at the surface. The hyphae were coenocytic with initially right-angled branching. Sporangia were globose or sub globose and papillated. Oogonia were smooth and globose (16.29-21.09 µm) in diameter. Antheridia were irregular, cylindrical and broadly attached to oogonia. Empty sporangia were also observed. Multilocus phylogenetic analysis using internal transcribed spacer region (Das et al. 2011), ß tubulin (Blair et al. 2008) and Cytochrome oxidase II gene (Noireung et al. 2020) showed that these isolates formed a stable clade with Phytopythium vexans (CBS119.80) sequence retrieved from NCBI database. BLAST analysis showed that ITS sequence of AP21 (OQ372986) and AP26 (OQ381083) had >99 % identity with P. vexans isolate NS-3 (ON533631). Further, BLAST analysis of ß tubulin (AP21 OQ446053, AP26 OR405377) and Cox II gene (AP21 OQ473414, AP26 OR552422) sequences showed that our Indian isolates showed >99 % similarity with P. vexans voucher strain CBS119.80. To fulfil Koch's postulates, Khasi mandarin (Citrus reticulata) seedlings were inoculated by adding 100 ml zoospore suspension of P. vexans (1x105 spores/ml) in sterilized soil (Thao et al. 2020). The experiment was carried out in triplicate. Yellowing of leaves and leaf drop were observed 7 days post inoculation while 30 days post inoculation, treated plants started showing symptoms of root rot, including mild root decay. No symptoms were observed in control treatment. The pathogen was reisolated from symptomatic roots and confirmed through colony and sporangium morphology. Recently, it was reported that P. vexans is associated with apple and pear decline in the Saiss plain of Morocco (Jabiri et al. 2021), root rot on mandarin in Thailand (Noireung et al. 2020) and on Durian in Vietnam (Thao et al. 2020). As per our knowledge, this is the first report of P. vexans causing root rot and gummosis in Khasi mandarin from north eastern states of India. This finding is significantly important for the development of a successful disease management strategy in India.

Comparative stress physiological analysis of aluminium stress tolerance of indigenous maize (Zea mays L.) cultivars of eastern Himalaya.

Yield potential of maize having distinct genetic diversity in Eastern Himalayan Region (EHR) hill ecologies is often limited by Al toxicity caused due to soil acidity. Stress physiological analysis of local check exposed to 0-300 µM Al under sand culture revealed that 150 µM Al as critical and 200 µM Al as tolerable limit. Increase in Al from 0 to 300 µM reduced total chlorophyll, carotenoids by 74.8 % and 44.7 % respectively and enhanced anthocyanin by 35.3 % whereas LA, SLW and SL have reduced by 81.3%, 21.3 % and 47.8 % respectively. R/S ratio was 51.0 and 13.7 % higher at lower Al levels (50 µM and 100 µM) and photosynthetic, transpiration rate and TDM were 62.5 %, 42.9 % and 78.6 % lower at higher Al (300 µM) as compared to control. TRL, RSA, RDW and RV at higher Al (300 µM) were 92.6 %, 98.7 %, 78.7 and 97.5 % lower over control respectively. Root and shoot Al and PUpE at higher Al (300 µM) was 194.0, 69.2 and 830 % higher whereas PUE decreased to 88.5 % over control. Evaluation of 31 indigenous maize cultivars at 0, 150, and 250 µM Al in sand culture, alongside tolerance scoring and assessment, revealed that Megha-9, Megha-10, and MZM-19 exhibits high Al tolerance, Megha-1, MZM-22, and MZM-42 demonstrated moderate tolerance, whereas Uruapara, Sublgarh, and BRL Para were identified as Al-sensitive. Stress physiological parameters like SDW, TDM, TRL, SL and LA contributed 46.02 % of variability to PC1, whereas A, RV, RSA, anthocyanin and Chlorophyll_b, contributed 13.56 % of variability to PC2. Highest values of CMS, SL, LP, LA, TRL and anthocyanin were recorded in cluster I having sensitive cultivars while highest CMS, SL, LA, LP, TRL and RSA were found in cluster II having moderately tolerant cultivars and highest mean values for TRL, RSA, LP, LA, CMS and SL were recorded in cluster III having highly Al stress tolerant cultivars. The traits viz., A, RV, RSA, anthocyanin and Chlorophyll_b, total chlorophyll and TDM were emanated as physio-morphological for assessing Al toxicity stress tolerance in Maize with high divergence values. Tolerant cultivars showing 63.4 % and 22.4 % higher anthocyanin at 150 µM Al and 250 µM Al than moderately tolerant one in acid soil experiment with increased root Al, shoot Al, root P and shoot P by 42.6 %, 11 %, 95.1 % and 34 % respectively were emerged as promising for novel maize improvement under acid soils of EHR.

A comparative study of localized phosphorus application and broadcasting method on biomass production and their use efficiency on Chilli (Capsicum annuum) under alkaline soil.

Rhizospheric based phosphorus (P) fertilizer management is necessary for crop production due to environmental concerns caused by the overuse of the broadcasting method and limited P reserves. This study proposes a comparison of P management that enhances P nutrition in Chilli (variety: Arka Khyati) through seedling root-dipping (SRD) in P-enriched slurry (SSP-amended; pH of 8.1), micro-dose placement (MDP; drill and place closer to plant root), and full dose (187.6 mg kg-1) placement by broadcasting (FD). In SRD, seedlings were dipped in five different P concentrations (50, 100, 200, 300, and 400 mg P2O5 kg-1) for varying durations (0, ½, 1, 2, 3, and 4 hours) and transplanted into pots (dipping in 0 mg P2O5 kg-1 consider as control), along with the MDP and FD treatments (total 33 treatments with 5 replications). [Seedlings dipped in 200, 300, and 400 mg P2O5 kg-1 died within a week after transplanting, thus were excluded from further analysis]. The amount of P received in MDP and FD were 21-90 times higher than P adhesion to seedling roots in SRD treatments. Root volume was in order SRD>MDP>FD. Seedlings dipped in 100 mg P2O5 kg-1 for 2 hours in SRD exhibited the highest biomass production, P-use and -recovery efficiency; and showed an increase of 52%, 178%, and 293% in FD, MDP, and SRD compared to the control respectively. It is recommended to use the SRD method with other P sources in reduced amount to maintain the native P pool in soil, and further multilocational trials are needed to validate.

Plant growth-promoting traits of culturable seed microbiome of citrus species from Purvanchal Himalaya.

Despite Northeastern India being "Treasure House of Citrus Genetic Wealth," genetic erosion of citrus diversity poses severe concern with a corresponding loss in seed microbial diversity. The seed microbiome of citrus species unique to the Purvanchal Himalaya is seldom explored for their use in sustainable orchard management. Isolation and characterization of culturable seed microbiomes of eight citrus species, namely, Citrus reticulata Blanco, C. grandis (L.) Osbeck, C. latipes Tanaka, C. megaloxycarpa Lushaigton, C. jambhiri Lush, C. sinensis (L.) Osbeck, C. macroptera Montr, and C. indica Tanaka collected from NE India were carried out. The isolates were then screened for an array of plant growth-promoting (PGP) traits [indole acetic acid (IAA) production, N2 fixation, phosphate and zinc complex dissolution, siderophores, and Hydrogen Cyanide (HCN) production]. The pure culture isolates of seed microbiomes were capable of dissolving insoluble Ca3(PO4)2 (1.31-4.84 µg Pi ml-1 h-1), Zn3(PO4)2 (2.44-3.16 µg Pi ml-1 h-1), AlPO4 (1.74-3.61 µg Pi ml-1 h-1), and FePO4 (1.54-4.61µg Pi ml-1 h-1), mineralized phytate (12.17-18.00 µg Pi ml-1 h-1) and produced IAA-like substances (4.8-187.29 µg ml-1 h-1). A few isolates of the seed microbiome were also able to fix nitrogen, secrete siderophore-like compounds and HCN, and dissolve ZnSO4 and ZnO. The 16S ribosomal Ribonucleic Acid (rRNA)-based taxonomic findings revealed that Bacillus was the most dominant genus among the isolates across citrus species. Isolates CG2-1, CME6-1, CME6-4, CME6-5, CME6-9, CJ7-1, CMA10-1, CI11-3, and CI11-4 were identified as promising bioinoculants for development of microbial consortium having multifaceted PGP traits for nutritional benefits of nitrogen, phosphorus and zinc, and IAA hormonal benefits to citrus crops for better fitness in acid soils.

Molecular interaction between plants and Trichoderma species against soil-borne plant pathogens.

Trichoderma spp. (Hypocreales) are used worldwide as a lucrative biocontrol agent. The interactions of Trichoderma spp. with host plants and pathogens at a molecular level are important in understanding the various mechanisms adopted by the fungus to attain a close relationship with their plant host through superior antifungal/antimicrobial activity. When working in synchrony, mycoparasitism, antibiosis, competition, and the induction of a systemic acquired resistance (SAR)-like response are considered key factors in deciding the biocontrol potential of Trichoderma. Sucrose-rich root exudates of the host plant attract Trichoderma. The soluble secretome of Trichoderma plays a significant role in attachment to and penetration and colonization of plant roots, as well as modulating the mycoparasitic and antibiosis activity of Trichoderma. This review aims to gather information on how Trichoderma interacts with host plants and its role as a biocontrol agent of soil-borne phytopathogens, and to give a comprehensive account of the diverse molecular aspects of this interaction.

Endophytic Beauveria bassiana can protect the rice plant from sheath blight of rice caused by Rhizoctonia solani and enhance plant growth parameters.

Beauveria bassiana, a potential entomopathogenic biocontrol agent, has recently drawn attention worldwide for its other additional beneficial roles such as plant disease antagonist, beneficial rhizosphere colonizer, plant growth promoter and an endophyte. In the present study, endophytic colonizing behaviour of five (5) B. bassiana isolates viz., Bb4, Bb16, Bb25, Bb44 and Bb53 were studied in rice following three (3) artificial inoculation techniques viz., seed treatment, root inoculation and foliar spray and the endophytic colonizing ability were determined by culture-based assay. After B. bassiana inoculation, rice plants were challenged with Rhizoctonia solani and disease incidence and plant growth promotion were assessed. Per cent colonization of rice stems, leaves and roots were influenced by inoculation technique, post-inoculation time (7th, 14th, 21st and 28th dpi) and plant growth medium (sterile soil, non-sterile soil), recorded maximum on 14th-day post-inoculation (dpi) i.e., 96% in stems, 92% in leaves and 28% in roots, whereas, lower colonization was recorded on 7th, 21st and 28th dpi. Whereas, the foliar spray was found best as compared to seed and root inoculation techniques, and maximum fungal recovery was observed in stems and leaves and least in roots. Upon colonization, the physical presence of B. bassiana in rice was localized by light microscopy-based studies. Potential B. bassiana strains with endophytic ability were re-isolated and their identity was determined based on morphometric and PCR-based techniques. Further, the present study also identified several virulent genes viz., BbChit1, Cdep1, Bbhog1 and Bbjen1 and extracellular hydrolytic enzymes viz., α-amylase, cellulase, lipase, pectinase and xylanase secreted by endophytic B. bassiana strains as determinants responsible for establishing the endophytic association in rice. On the other hand, a significant reduction in disease incidence was observed in the endophytic B. bassiana Bb4-, Bb16- and Bb44-inoculated plants as compared to the non-endophytic B. bassiana Bb25- and Bb53-inoculated plants along with enhanced plant growth promotion. This is one of the few studies investigating the colonization of B. bassiana in rice and its promising role as a plant disease antagonist and plant growth promoter in rice.

Linking phylogeny to abundant ribotypes of community fingerprints: an exercise on the phylotypic responses to plant species, fertilisation and Lolium perenne ingression.

The present study explores the potential of directly linking phylogenetic identities obtained by cloning and sequencing of ITS sequences to dominant ribotypes of molecular community fingerprints to give further insight into dominant members of the communities in three Irish grassland soils. The ten most abundant bacterial ribotypes of untreated bare soils of three grassland microcosms were chosen to represent the "baseline community" of the respective soil. Identities on phylum and order level were assigned to these ribotypes on a weighted basis, by matching sequence homologies of cloned ITS sequences with ribotypes of the same fragment lengths ±5 bp. Results showed that ribotypes were represented by the phyla Acidobacteria, Actinobacteria, Proteobacteria, and Firmicutes and the distribution of the ribotype and phylotype communities was shown to be highly site-specific. Furthermore the response of dominant bacterial phylotypes to plant species composition, fertilisation and Lolium perenne ingression was investigated within a larger microcosm study (Microb Ecol 63:509-521).

Influences of plant species composition, fertilisation and Lolium perenne ingression on soil microbial community structure in three Irish grasslands.

Semi-natural grassland soils are frequently fertilised for agricultural improvement. This practice often comes at a loss of the indigenous flora while fast-growing nitrogen-responsive species, such as Lolium perenne, take over. Since soil microbial communities depend on plant root exudates for carbon and nitrogen sources, this shift in vegetation is thought to influence soil microbial community structure. In this study, we investigated the influence of different plant species, fertilisation and L. perenne ingression on microbial communities in soils from three semi-natural Irish grasslands. Bacterial and fungal community compositions were determined by automated ribosomal intergenic spacer analysis, and community changes were linked to environmental factors by multivariate statistical analysis. Soil type had a strong effect on bacterial and fungal communities, mainly correlated to soil pH, as well as soil carbon and nitrogen status. Within each soil type, plant species composition was the main influencing factor followed by nitrogen fertilisation and finally Lolium ingression in the acidic upland and mesotrophic grassland. In the alkaline grassland, however, Lolium ingression had a stronger effect than fertilisation. Our results suggest that a change in plant species diversity strongly influences the microbial community structure, which may subsequently lead to significant changes in ecosystem functioning.

Gut wall bacteria of earthworms: a natural selection process.

Earthworms and microorganisms are interdependent and their interactions regulate the biogeochemistry of terrestrial soils. Investigating earthworm-microorganism interactions, we tested the hypothesis that differences in burrowing and feeding habits of anecic and endogeic earthworms are reflected by the existence of ecological group-specific gut wall bacterial communities. Bacterial community was detected using automated ribosomal intergenic spacer analysis of 16S and 23S genes and ribotype data was used to assess diversity and community composition. Using soil and earthworm samples collected from adjacent wheat-barley and grass-clover fields, we found that the anecic Lumbricus terrestris and L. friendi, the endogeic Aporrectodea caliginosa and A. longa (classically defined as anecic, but now known to possess endogeic characteristics) contain ecological group-specific gut wall-associated bacterial communities. The abundance of specific gut wall-associated bacteria (identified by sequence analysis of ribotype bands), including Proteobacteria, Firmicutes and an actinobacterium, was ecological group dependent. A microcosm study, conducted using A. caliginosa and L. terrestris and five different feeding regimes, indicated that food resource can cause shifts in gut wall-associated bacterial community, but the magnitude of these shifts did not obscure the delineation between ecological group specificity. Using A. caliginosa and A. longa samples collected in six different arable fields, we deduced that, within an ecological group, habitat was a more important determinant of gut wall-associated bacterial community composition than was host species. Hence, we conclude that the selection of bacteria associated with the gut wall of earthworms is a natural selection process and the strongest determinant of this process is in the order ecological group>habitat>species.

Field preservation and DNA extraction methods for intestinal microbial diversity analysis in earthworms.

We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based on a sodium dodecyl sulphate (SDS) buffer. For both of these buffers, incorporation of a bead-beating during the lysis step increased the ARISA-derived bacterial ribotype numbers and diversity estimates, as determined for gut wall samples (P<0.01). Although spectrophotometric analysis indicated that DNA extracted by the CTAB-DTT and SDS-based methods were of comparable quality (P> or =0.05), the former method yielded >1.5 times more DNA from both gut contents and gut walls of earthworms than the latter method (both incorporating the bead beating step) (P<0.01). ARISA analysis detected more reproducible ribotypes and more microbial diversity in DNA extracted by the CTAB-DTT- as compared to the SDS-based method (P<0.01). Significant difference between bacterial communities of gut contents and gut walls were detected within DNA extracted by the CTAB-DTT (but not by the SDS-based) method (Global R=0.76, P<0.001, analysis of similarity). Using the CTAB-DTT-based method, we showed that earthworm preservation in ethanol yielded higher quality DNA from gut contents than preservation in either chloroform or liquid N, as determined by spectrophotometry, PCR inhibition analysis and bacterial and fungal ARISA (P<0.05). Bacterial or fungal communities in the gut contents of fresh and ethanol-preserved earthworms were more similar and were significantly different from those of earthworms preserved in chloroform or liquid N (Global R=0.79 and 0.83 for bacteria and fungi, respectively; P<0.001, analysis of similarity). We propose that ethanol preservation and the CTAB-DTT-based DNA extraction method described herein are also suitable for the analysis of gut-associated microbiota in other soil and sediment feeding invertebrates.