16S rDNA Sequence Analysis Research Articles

Sequence analysis of 16S rDNA, gyrB and alkB genes of plant-associated Rhodococcus species from Tunisia

The genus Rhodococcus contains several species with agricultural, biotechnological and ecological importance. Within this genus, many phyllosphere, rhizosphere and endosphere strains are plant growth promoting bacteria, whereas strains designated as R. fascians are plant pathogens. In this study, we isolated 47 Rhodococcus strains from a range of herbaceous and woody plant species. Phylogenetic analysis based on 16S rDNA, gyrB and alkB genes was used to compare our strains with type strains of Rhodococcus. For most of our strains, sequence similarity of the 16S rDNA, gyrB and alkB regions to type strains ranged from 98-100 %. Results of the concatenated gene sequence comparisons identified 18 strains of R. fascians and three strains of R. kroppenstedtii. The remaining strains were unclassified, and may represent novel species of Rhodococcus. Phylogenetic analysis based on gyrB sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis of alkB sequences was unable to identify strains with orange-coloured colonies to species level.

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize (“Rojo Criollo”) and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and β-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.

Isolation and identification of herbivorous ciliates from contaminated microalgal cultures

Ciliates are a common but understudied group of grazers that can invade microalgal cultures. To estimate the potential impact of ciliates on microalgal culture productivity, the identification of species that can invade these cultures is essential. Furthermore, isolation of these herbivorous ciliates allows to use them in experiments that investigate the impact of ciliate grazing on the productivity of microalgal cultures. The main aims of this study were to isolate and identify ciliates that invade cultures of the freshwater microalgae Chlorella and Chlamydomonas, and to establish a live collection of these ciliates for usage in future experiments. To this end, we optimized a method for isolating ciliates from contaminated microalgal cultures and we developed a new PCR primer set for amplifying the partial 18S rDNA of ciliates belonging to the classes Spirotrichea, Oligohymenophorea and Colpodea. As a result, we isolated 11 ciliates from microalgal enrichment cultures inoculated with non-sterile dust and various freshwater sources. Of these 11 species, 7 were found to be feeding on Chlamydomonas. Ciliate species that fed on Chlorella could not be isolated in this study. Ciliate species feeding on Chlamydomonas were identified based on a combination of morphological observations and molecular analyses of partial 18S rDNA sequences.

Efficiency of potassium-solubilizing Paenibacillus mucilaginosus for the growth of apple seedling

Chemical potassium (K) fertilizer is commonly used in apple (Malus domestica L. Borkh) production but K is easily fixed by soil, resulting in reduced K fertilizer utilization and wasted resources. K-solubilizing bacteria (KSB) can cost-effectively increase the soluble K content in rhizosphere soil. Therefore, the objectives were to select high-efficiency KSB from apple orchards under various soil management models and evaluate their effects on apple seedling growth. Maize (Zea mays L.) straw mulching (MSM) increased the total carbon (TC), total nitrogen (TN) and available potassium (AK) in the rhizosphere and improved fruit quality. The number of KSB in the rhizosphere soil of MSM was 9.5×104 CFU g−1 soil, which was considerably higher than that in the other mulching models. Fourteen KSB strains were isolated with relative K solubilizing ability ranging from 17 to 30%, and five strains increased the dry weight per apple seedling. The most efficient strain was identified as Paenibacillus mucilaginosus through morphological observation and sequence analysis of 16S rDNA, named JGK. After inoculation, the colonization of JGK in soil decreased from 4.0 to 1.5×109 CFU g−1 soil within 28 d. The growth of the apple seedlings and the K accumulation in apple plants were promoted by irrigation with 50 mL JGK bacterial solution (1×109 CFU mL−1), but there was no significant increase in the AK content of rhizosphere soil. High-performance liquid phase analysis (HPLC) data showed that the JGK metabolites contained phytohormones and organic acids. Hence, the JGK strain promoted the growth of two-month-old apple seedlings by stimulating function of the produced phytohormones and enhanced K solubility by acidification for apple seedling uptake. This study enriches the understanding of KSB and provides an effective means to increase the K utilization efficiency of apple production.

Genetic diversity of phytoplasma strains infecting chrysanthemum varieties in India and their possible natural reservoirs

Symptoms typical of phytoplasma infection such as phyllody, virescence, witches’ broom and yellowing were observed in 12 varieties of Chrysanthemum morifolium in floral nurseries and experimental fields at New Delhi, Karnataka, Maharashtra and Andhra Pradesh, India, during surveys made from 2015 to 2017. Disease incidence ranged from 15 to 30%. Phytoplasma presence was confirmed in all symptomatic chrysanthemum varieties by molecular identification assays. Sequence comparison, phylogenetic and in silico RFLP analyses of 16S rDNA sequences allowed the identification of the chrysanthemum infecting phytoplasma strains into different ribosomal groups and subgroups, namely 16SrI, 16SrII-D, 16SrVI-D and 16SrXIV. Detection of phytoplasma strains of 16SrII-D subgroup were also confirmed in symptomatic Chenopodium album and Parthenium hysterophorus plants grown in and around the surveyed chrysanthemum fields at New Delhi, whereas 16SrVI-D phytoplasma strains were detected in symptomatic Cannabis sativa weed and leafhopper Hishimonus phycitis individuals collected from the symptomatic chrysanthemum fields at New Delhi. This is the first report on the presence of 16SrVI and 16SrXIV groups of phytoplasmas in chrysanthemum plants. Studies on genetic diversity of phytoplasmas infecting the major chrysanthemum varieties in India and their epidemiological aspects had previously not been reported. The detection and identification of phytoplasmas in different chrysanthemum varieties could contribute to increase the awareness among farmers in the management of these diseases.

Morphological and molecular characterization of Cystoisospora laidlawi oocysts (Apicomplexa: Eimeriidae) in farmed American mink (Neovison vison) in Denmark.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5μm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

세포외 다당류를 생성하는 유산균 Weissella cibaria JNU29의 분리 및 특성과 이를 이용한 한국전통 설기떡 제조

Due to the nutritional value of rice cakes, there is renewed interest in this traditional Korean food, and thus various efforts have been made to improve its storage stability. In this study, lactic acid bacteria that produce exopolysaccharides (EPS) were isolated and their physiochemical properties were analyzed. The bacteria were used to produce ‘Sulgidduk’ (a Korean rice cake) with improved storage stability. Among the 107 strains isolated from traditional fermented foods, only five strains were confirmed to produce EPS in MRS media with 10% sucrose. Two of these strains were identified as members of the genus Weissella and three were identified as the Bacillus genus via sequence analysis of 16S rDNA. Among the five strains, the strain Weissella cibaria JNU29 was selected for application to ‘Sulgidduk’, since it is food applicable, viable in artificial gastric and bile juices, and produces the highest content of EPS (16.72 g/L). TLC and HPLC analysis showed that the EPS produced by W. cibaria JNU29 strain was a homopolysaccharide composed of only glucose. A culture solution of the W. cibaria JNU29 strain was mixed with milled rice flour to prepare ‘Sulgidduk’. After incubation for 48 h, the ‘Sulgidduk’ showed lower hardness values than that of the control without the culture solution, and the lactic acid bacteria survived to a level of 7 log CFU/g. Thus, the W. cibaria JNU29 strain, which produces EPS, can be applied for the production of ‘Sulgidduk’, as it delays retrogradation and prevents mold-induced decay to improve shelf life.

Lactobacillus reuteri Ameliorates Intestinal Inflammation and Modulates Gut Microbiota and Metabolic Disorders in Dextran Sulfate Sodium-Induced Colitis in Mice.

Lactobacillus reuteri, a commensal intestinal bacteria, has various health benefits including the regulation of immunity and intestinal microbiota. We examined whether L. reuteri I5007 could protect mice against colitis in ameliorating inflammation, modulating microbiota, and metabolic composition. In vitro, HT-29 cells were cultured with L. reuteri I5007 or lipopolysaccharide treatment under three different conditions, i.e., pre-, co- (simultaneous), and posttreatment. Pretreatment with L. reuteri I5007 effectively relieves inflammation in HT-29 cells challenged with lipopolysaccharide. In vivo, mice were given L. reuteri I5007 by gavage throughout the study, starting one week prior to dextran sulfate sodium (DSS) treatment for one week followed by two days without DSS. L. reuteri I5007 improved DSS-induced colitis, which was confirmed by reduced weight loss, colon length shortening, and histopathological damage, restored the mucus layer, as well as reduced pro-inflammatory cytokines levels. Analysis of 16S rDNA sequences and metabolome demonstrates that L. reuteri I5007 significantly alters colonic microbiota and metabolic structural and functional composition. Overall, the results demonstrate that L. reuteri I5007 pretreatment could effectively alleviate intestinal inflammation by regulating immune responses and altering the composition of gut microbiota structure and function, as well as improving metabolic disorders in mice with colitis.

Molecular characterization of B. anthracis isolates from the anthrax outbreak among cattle in Karnataka, India

BackgroundAnthrax, a zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis. During January 2013, an anthrax outbreak among cattle was reported in Gundlupet Taluk, neighboring Bandipur National Park and tiger reserve, India. The present study aims at the molecular identification and characterization of 12 B. anthracis isolates from this outbreak by 16S rRNA gene sequencing, screening B. anthracis specific prophages and chromosomal markers, protective antigen (pag) gene and canonical single nucleotide polymorphism (canSNP) analysis to subtype the isolates into one of the twelve globally identified clonal sub-lineages of B. anthracis.ResultsThese isolates had identical 16S rDNA nucleotide sequences with B. anthracis specific dual peaks showing mixed base pair R (G/A) at position 1139 with visual inspection while the automated basecaller software indicated a G. Alternatively the nucleotide A at 1146 position was indicative of the 16S rDNA type 7. Multiple sequence alignment with additional 170 (16S rDNA) sequences of B. cereus sensu lato group from GenBank database revealed 28 new 16S types in addition to eleven 16S types reported earlier. The twelve B. anthracis isolates were found to harbor the four B. anthracis specific prophages (lambdaBa01, lambdaBa02, lambdaBa03, and lambdaBa04) along with its four specific loci markers (dhp 61.183, dhp 77.002, dhp 73.019, and dhp 73.017). The pag gene sequencing identified the isolates as protective antigen (PA) genotype I with phenylalanine-proline-alanine phenotype (FPA phenotype). However, sequence clustering with additional 34 pag sequences from GenBank revealed two additional missense mutations at nucleotide positions 196 bp and 869 bp of the 2294 bp pag sequence among the 5 B. cereus strains with pXO1 like plasmids. The canSNP analysis showed that the isolates belong to A.Br.Aust94 sub-lineage that is distributed geographically in countries of Asia, Africa, Europe and Australia.ConclusionsThe analysis of 16S rDNA sequences reiterated the earlier findings that visual inspection of electropherogram for position 1139 having nucleotide R could be used for B. anthracis identification and not the consensus sequence from base caller. The canSNP results indicated that the anthrax outbreak among cattle was caused by B. anthracis of A.Br.Aust94 sub-lineage.

Etiology of potato soft rot and blackleg diseases complex in Egypt

Recent observations of increasing incidences of potato plants showing blackleg symptoms have caused serious concern for potato growers and traders in Egypt. Potato plants showing blackleg and soft rot symptoms were sampled from main potato-growing areas in Egypt during three successive seasons. Dickeya spp., Pectobacterium carotovorum and Pectobacterium atrosepticum were successfully isolated and identified from symptomatic potato plants (tubers and stems). The identity of bacterial isolates was reviled by identification methods based on different biological principles: cultural, biochemical and molecular methods. Specific primers targeting different genomic loci were selected for molecular identification, utilizing either conventional or real-time PCR. Multiple species were isolated from some infected samples revealing complex infection, which confirm the complexity and interaction of soft rot and blackleg diseases. Given that no previous reports on Dickeya diseases in Egypt were confirmed since the recent revision of its taxonomy, the result of PCR with Dickeya spp. was furtherly confirmed using DNA sequencing assay. BLASTn analyses of 16S rDNA sequences showed similarity to several species of Dickeya, with 99% nucleotide similarity to strains of, D. chrysanthemi, D. dianthicola and D. solani, respectively. Furthermore, the most commonly identified bacterial pathogen in the tested samples was P. carotovorum followed by P. atrosepticum and Dickeya spp., while P. brasiliense was never detected in any of the collected samples confirmed by real-time PCR assay. The results of such field survey suggested to help the decision makers in Egypt to establish an action plan to face the increasing risk of potato diseases caused by Dickeya spp. for the benefit of Egyptian domestic production.

Two new species of a new genus, Pseudorogneda, of Polycystididae discovered in Southern China.

Two new species of the new genus Pseudorogneda of Polycystididae were discovered in a brackish environment from Southern China. Pseudorogneda sinensis n. gen. n. sp., is distinguished by a semicircular double-walled prostate stylet type II that tapers in diameter from proximal to distal with a proximal-to-distal fold, and an inner wall with a funnel-shaped base originating at the midpoint of the outer wall; its flattened tubular prostate stylet type IV exhibits a horn-like base and a proximal-to-distal folded structure on its wall. For Pseudorogneda shenda n. gen. n. sp., its crescent-shaped prostate stylet type II is double-walled and the inner wall has a funnel-shaped base that starts at the proximal 2/3 position of the outer stylet and is fused with the outer stylet to form a sharp distal end; its tubular and slightly-curved prostate stylet type IV exhibits a slit and a distal fold. Phylogenetic analysis of 18S rDNA and 28S rDNA sequences shows that the two novel species are clustered to form an independent clade from other genera, which is consistent with morphological comparison in the establishment of two new species in a new genus.

Preliminary Characterization of the First Phosphate Solubilizing Bacteria Isolated From the Rhizospheric Soil of the Jujube Plant Ziziphus

The use of rhizobacteria that solubilize inorganic phosphates as biofertilizers is considered as a suitable solution for sustainable agriculture. Only limited number of studies have been carried out on the jujube plant Ziziphus, which is frequently encountered the Mediterranean basin, particularly in North Africa. Here, we conducted the first study on jujube to characterize the native phosphate solubilizing bacteria (PSB) from its rhizospheric soil. jujube was selected because of its ability to survive within arid climates by tolerating biotic and abiotic stresses. A total of 41 rhizobacteria isolated from rhizospheric jujube collected from the experimental farm of Benguerir (Morocco) were able to solubilize tri-calcium phosphate (TCP) on NBRIP agar. Next, thirteen of the best performing isolates were subsequently tested for phosphate solubilization in liquid media and for antibiotics resistance. Eleven exhibit a great ability to release soluble phosphate from TCP with ranged concentrations between minima at 160 mg/L and maxima at 260 mg/L. Moreover, several PSB strains confer resistance at least to one of the tested antibiotics. Analysis of 16S rDNA sequencing demonstrated that PSB isolates were closely related to the genus Pseudomonas, Bacillus, Enterobacter, or Paenibacillus.

Mussaenda erythrophylla: A new host of ‘Candidatus Phytoplasma asteris’ in India

Leaf yellowing symptoms were observed in Mussaenda erythrophylla at Digiyana Park, Jammu during June 2016. Leaf yellowing and flat stem symptoms were also observed on nearby planted Catharanthus roseus and Celosia argentea, respectively. Amplicons of ~ 1.25 kb were consistently amplified in nested PCR assays with DNA extracted from all the symptomatic Mussaenda erythrophylla, Catharanthus roseus and Celosia argentea samples using primer pairs P1/P7 followed by R16F2n/R2. However, no amplifications were achieved with any of the asymptomatic plant samples. Pair wise sequence comparison of the ~ 1.25 kb products of 16S rDNA sequence of M. erythrophylla leaf yellowing, C. roseus leaf yellowing and C. argentea flat stem phytoplasma isolates revealed 99.8% sequence identity with ‘Candidatus Phytoplasma asteris’ related strains. Phylogeny and virtual RFLP analysis of 16S rDNA sequences confirmed the affiliation of ‘Ca. P. asteris’ subgroup I-B with all the three symptomatic plant samples. This is the first report of association of ‘Ca. P. asteris’ subgroup I-B with M. erythrophylla worldwide.

Isolation and characteristics of multi-drug resistant Streptococcus porcinus from the vaginal secretions of sow with endometritis

BackgroundSow endometritis is a common disease in pig breeding farms after artificial insemination, which leads to gray-green vaginal secretions and decreased conception rates. It is important to perform an etiologic diagnosis for effective treatments and control of diseases. The aim of this study was to carry out a pathogenic detection in five specimens of vaginal secretions collected from sick pigs with endometritis, implement identification of the pathogens by phenotypic detection and 16 s rDNA sequence and phylogeny analysis, and determinate antibiotic susceptibility of the isolates.ResultsA Streptococcus strain was isolated and identified from all of the five specimens. The isolate was positive for Voges-Proskauer (V-P) and for the hydrolysis of arginine, esculin and myelin-associated glycoprotein (MAG). Acid formation was observed for sorbitol, mushroom sugar, sucrose, and glucose. The 16S rDNA sequence of the isolate possessed 99.93% similarity to that of Streptococcus porcinus. The phylogenetic analysis of 16S rDNA sequence showed that the isolate belonged to the same clade as the S. porcinus strains from humans, pigs, and other animals. The isolate exhibited multi-drug resistance to aminoglycosides, quinolones, macrolides and tetracyclines except being sensitive to some β- lactams such as penicillin G, cephalothin, cefazolin, cephradine and cefuroxime.ConclusionsA S. porcinus isolate with multi-drug resistance was identified from vaginal secretions of sows with endometritis in one pig breeding farm, which suggests that the sow endometritis was caused by S. porcinus infection during artificial insemination. This study indicates that sensitive antibiotics such as penicillin G or some cephalosporins could be used for treatment of the diseases. In addition, the study hints that bacterial multi-drug resistance is a tough problem for disease treatment in pig farms.

Isolation and characterization of Bacillus subtilis strain 1-L-29, an endophytic bacteria from Camellia oleifera with antimicrobial activity and efficient plant-root colonization.

Endophytic bacteria, which are common in plant tissues, may help to control plant pathogens and enhance plant growth. Camellia oleifera, an oil-producing plant, is widely grown in warm, subtropical, hilly regions in China. However, C. oleifera is strongly negatively affected by C. oleifera anthracnose, which is caused by Colletetrichum fructicola. To find a suitable biocontrol agent for C. oleifera anthracnose, 41 endophytes were isolated from the stems, leaves, and roots of C. oleifera. Bacterial cultures were identified based on analyses of 16S rDNA sequences; most strains belonged to the genus Bacillus. The antagonistic effects of these strains on C. fructicola were tested in vitro. In total, 16 strains inhibited C. fructicola growth, with B. subtilis strain 1-L-29 being the most efficient. Strain 1-L-29 demonstrated antagonistic activity against C. siamense, C. asianum, Fusarium proliferatum, Agaricodochium camellia, and Pseudomonas syringae. In addition, this strain produced indole acetic acid, solubilized phosphate, grew on N-free media, and produced siderophores. To facilitate further microecological studies of this strain, a rifampicin-resistant, green fluorescent protein (GFP)-labeled strain, 1-L-29gfpr, was created using protoplast transformation. This plasmid had good segregational stability. Strain 1-L-29gfpr was re-introduced into C. oleifera and successfully colonized root, stem, and leaf tissues. This strain remained at a stable concentration in the root more than 20 d after inoculation. Fluorescence microscopic analysis showed that strain 1-L-29gfpr thoroughly colonized the root surfaces of C. fructicola as well as the root vascular tissues of Arabidopsis thaliana.

Biodegradation of penicillin G from industrial bacteria residue by immobilized cells of Paracoccus sp. KDSPL-02 through continuous expanded bed adsorption bioreactor

BackgroundAn efficient biodegradation-strengthening approach was developed to improve penicillin G degradation from industrial bacterial residue in an expanded bed adsorption bioreactor (EBAB) is reported in this paper.ResultsParacoccus sp. strain KDSPL-02 was isolated based on its ability to use penicillin G as the sole carbon and nitrogen source. Strain identification was based on analyses of morphology, physio-biochemical characteristics, and 16S rDNA sequences. The effects of temperature, pH, PVA-sodium alginate concentration, calcium chloride concentration and initial penicillin G concentration were investigated. Repeated operations of immobilized cells with EBAB, At initial penicillin concentrations below 2.0 g L− 1, the continuous mode could reach more than 20 times, and the degradation rate reached 100%.ConclusionsThe present study suggests that the EBAB system can be utilized for the simple and economical biodegradation of penicillin G from industrial bacterial residue.

Burkholderia gladioli causes bacterial internal browning in sweetpotato of China

A new bacterial disease of sweetpotato was recently identified in Puning, Guangdong Province, China and is referred to bacterial internal browning of sweetpotato in this paper. One-third of the tubers of sweetpotato were infected in the field. These tubers in the field with bacterial inner brown displayed yellowing on the skin that turned into slightly roughened and cracked surface with progress in disease. Cross-sections of these roots revealed a yellowish brown or dark brown circle in the vascular cambium. Pathogenicity tests were conducted on different organs of two sweetpotato cultivars and revealed that the isolated bacteria are pathogenic on stems and roots. We confirmed the causal agent as Burkholderia gladioli based on biochemical and physiological characterization, carbon source utilization, and sequence analysis of partial 16S rDNA and 16S–23S rDNA internal transcribed spacer (ITS) region. To our knowledge, it is the first report on the occurrence of B. gladioli in sweetpotato as a pathogen.

Biocontrol potential of Pseudomonas azotoformans, Serratia marcescens and Trichoderma virens against Fusarium wilt of Dalbergia sissoo

AbstractWilt disease caused by Fusarium solani is a serious constraint to Dalbergia sissoo (shisham) plantations in northern India. In this study, the antagonistic potential of 40 bacterial isolates recovered from rhizophere soil of healthy shisham trees, and a well‐characterized Trichoderma species (Trichoderma virens) were tested for their possibility as biocontrol agents for F. solani. Two promising isolates (S1 and S15) were identified which inhibited pathogen growth, caused chitin degradation, produced siderophores and solubilized phosphate in vitro. Isolate S15 scored highest for hydrogen cyanide (HCN) production while isolate S1 was a non‐HCN producer. These two isolates were identified as Serratia marcescens (S1) and Pseudomonas azotoformans (S15) following sequence analysis of 16S rDNA. In dual culture assays, T. virens caused 80% inhibition of mycelial growth of the test fungus. The three selected antagonists when tested in planta in the glasshouse completely suppressed production of wilt symptoms on 12‐month‐old shisham plants. Further work is needed to ascertain the potential of these isolates to be used as biocontrol agents to manage shisham wilt under field conditions.

First Report of a ‘Candidatus Phytoplasma aurantifolia’ Strain Associated with Virescence, Floral Proliferation, and Dwarf Symptoms on Indigofera suffruticosa in Taiwan

Indigofera suffruticosa Mill., generally known as anil or Guatemalan indigo, is a perennial shrub planting in tropical and subtropical regions, including Taiwan; it was commonly used to produce the indigo dye, which is still being used in many personal workshops in Taiwan. There were a few reported cases of the association of phytoplasma with Indigofera spp. In 1983, I. suffruticosa showing phyllody and small leaf symptoms was first described, but no molecular identification was conducted (Albanese et al. 1995). Indigofera linifolia with little leaf, phyllody, and proliferation symptoms and Indigofera colutea showing little leaf symptoms in Australia caused by phytoplasma strains were reportedly associated with papaya yellow crinkle disease (Padovan and Gibb 2001). Affected Indigofera juncea and Indigofera hirsuta exhibiting witches’-broom symptoms were reported to be the same as the sesame phyllody phytoplasma isolate in Thailand (Nakashima et al. 1995). In February 2016, I. suffruticosa plants showing virescence, floral proliferation, and dwarf symptoms were collected from commercial fields of Mingjian Township, Nantou County, Taiwan. Two each of symptomatic and asymptomatic samples were collected from the field for further study. Transmission electron microscopic observation revealed the presence of the pleomorphic phytoplasma cells only in the phloem sieve cells of the two symptomatic I. suffruticosa plants. The two asymptomatic and two symptomatic samples were used for total DNA extraction with the modified CTAB method, and the amplicons of the nested PCR with universal primer pairs for nearly full-length 16S rDNA amplification (P1/P7 followed by R16F2n/R16R2) were electrophoresed in 0.8% agarose gel (Fulton et al. 1995; Lee et al. 1998). A 1.2-kb fragment (GenBank accession no. KX268346) was identified only from the two symptomatic I. suffruticosa plants. Analysis of 16S rDNA sequences by iPhyClassifier indicated that the phytoplasma associated with virescence symptoms in I. suffruticosa shared a 98.2% identity with that of the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession no. U15442). Virtual restriction fragment length polymorphism patterns were identical to those of the 16SrII-V subgroup (Zhao et al. 2009). Taken together, the results indicated that the phytoplasma associated with I. suffruticosa in Taiwan is a ‘Ca. P. aurantifolia’-related strain. The occurrence of phytoplasma in I. suffruticosa could have direct implication on economic profit for growers and the handicraft industry in Taiwan. To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma causing virescence, floral proliferation, and dwarf symptoms on I. suffruticosa in Taiwan.

Cannabis sativa L.: A Potential Natural Reservoir of Sugarcane Grassy Shoot Phytoplasmas in India

Cannabis sativa plants growing nearby sugarcane fields affected with grassy shoot disease at Muzaffarnagar, Uttar Pradesh, India, showed severe witches’ broom symptoms. The DNA from symptomatic Cannabis and sugarcane samples was isolated and subjected to nested PCR assays with universal phytoplasma-specific primer pairs (P1/P7 and R16F2n/R16R2). A DNA fragment of ~ 1.2 kbp was amplified in all the symptomatic samples of sugarcane and Cannabis with R16F2n/R16R2 primer pair but was absent in non-symptomatic samples. Pairwise sequence comparison, phylogeny and in silico RFLP analysis of 16S rDNA sequences confirmed the identity of a rice yellow dwarf phytoplasma group-related strain (16SrXI-B) associated with Cannabis which suggested its role as potential natural reservoir of SCGS phytoplasma in nature.