16S rDNA-RFLP Research Articles

Study of Phosphate Solubilizing Fluorescent Pseudomonas Recovered from Rhizosphere and Endorhizosphere of Aloe barbadensis (L.)

Phosphorus (P) is a key nutrient required by plants for their growth and development. A large part of applied phosphatic fertilizer becomes unavailable due to its fixation in soil. In rhizosphere, fluorescent Pseudomonads are instrumental in phosphate solubilization. We isolated Pseudomonads from the rhizosphere of Aloe barbadensis (L.), and screened them for their plant growth promoting ability such as - solubilization of phosphate, auxin production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. A total of 307 fluorescent Pseudomonas were isolated and screened, of which, 24 isolates showed a strong phosphate solubilizing ability, ranging from 69.71 to 236.73 µg ml−1 . The Indole acetic acid (IAA) production by all 24 isolates varied from 28.44 to 151.99 µg ml−1. However, 17 isolates produced ACC-deaminase ranging from 8.19 to 10.27 µmol mg−1 h−1. Analyses of 16S rRNA gene and Pseudomonas-specific genes grouped the isolates in seven different species of fluorescent Pseudomonas. Genotypic analysis based on 16S rDNA-RFLP using restriction enzymes (HaeIII, AluI and MspI) and BOX-PCR generated unique genotype specific pattern. The results suggested that Pseudomonas diversity has no association with microenvironments and the sampling site of the plants. 16S rRNA gene-based diversity indices revealed great diversity among the fluorescent Pseudomonas recovered from the rhizospheric regions of the Aloe. Due to the above plant growth-promoting traits, fluorescent Pseudomonas can be exploited as bio-inoculants for crops, with minimal damage to the environment.

First report of ‘Candidatus Phytoplasma australasia’‐related strains associated with disease of Jasminum multiflorum in India

Jasminum multiflorum (cv. Star Jasmine; family Oleaceae) is an important ornamental shrub native to South-East Asia. It is cultivated across the tropics for its ornamental value, in particular its fragrant, beautiful white flowers. During February 2020, symptoms of leaf yellowing and the premature decline of inflorescences were observed on 6.5% of plants grown in home gardens (Figs. 1, 2) in the Uttara Kannada district of Karnataka state, India. Four symptomatic and two symptomless plants were subjected to genomic DNA extraction using the CTAB method. Nested PCR was performed with universal phytoplasma 16S rRNA primers P1/P7 (Deng & Hiruki, 2; Schneider et al., 6) and R16F2n/R16R2 (Gundersen & Lee, 4). A DNA extract from the periwinkle virescence phytoplasma of group 16SrI, ‘Candidatus Phytoplasma asteris’ (MN223677) was used as the positive control. Amplification of the 16S rRNA gene was obtained only from symptom-bearing jasmine and the positive control, but not from the symptomless plants. The nested PCR products (~1.25kb) were purified and sequenced directly. Sequences were compared to those of reference phytoplasmas and subjected to in silico RFLP with iPhyclassifier (Zhao et al., 2009) and phylogenetic analysis using the neighbour-joining method of MEGA X (Kumar et al., 2018). The R16F2n/R16R2 gene sequences of the J. multiflorum phytoplasmas (GenBank Accession Nos. MW049328-MW049329) showed 99.9-100% sequence identity with those of the reference phytoplasmas of the former Peanut witches’ broom (16SrII) group, ‘Candidatus Phytoplasma aurantifolia’, including ‘Ca. P. australasia’ (Y10097). The virtual RFLP profiles of both J. multiflorum phytoplasmas were identical to each other and to those of the ‘Ca. P. australasia’ related strain (Y10097) of the 16SrII-D subgroup with a similarity coefficient of 1.00. The phylogenetic tree confirmed that the J. multiflorum phytoplasmas were closely related to the 16SrII-D, ‘Ca. P. australasia’ strain cluster (Fig. 3). Based on the results of the 16S rDNA nucleotide sequence comparison, phylogenetic and RFLP analyses, the J. multiflorum phytoplasmas associated with inflorescence yellowing and premature decline were identified as strains of the ‘Ca. P. australasia’, 16SrII-D subgroup. Phytoplasmas of 16SrII, 16SrXI (‘Ca. P. oryzae’) and 16SrI (‘Ca. P. asteris’) groups have been reported previously affecting J. sambac in Oman and India (Al-Zadjali et al., 1; Madhupriya et al., 5; Gopala & Rao, 3). However, this is the first report of the association of ‘Ca. P. australasia’ affecting J. multiflorum in India. Considering the economic importance of jasmine for the ornamental industry, this report provides significant information to support informed phytosanitary measures to prevent the spread of ‘Ca. P. australasia’ to nearby jasmine and other important members of the Oleaceae. The authors thank Department of Science and Technology, Government of India for providing financial assistance through DST-INSPIRE Fellowship scheme (IF.160687).

Symbiotic, phenotypic and genotypic characterization of Bradyrhizobium sp. nodulating Spartium junceum L. from Bejaia, northeastern Algeria

Twenty-three bacterial strains were isolated from root nodules of Spartium junceum L., growing wild in four distinct geographic locations in Bejaia city (Northeastern Algeria). They were analyzed based on their symbiotic effectiveness, phenotypic properties and restriction fragment length polymorphism of the 16S rRNA gene. In addition, sequence analyses of the ribosomal gene, protein-coding housekeeping genes glnII and recA, and symbiotic gene (nodC) were used to clarifying their taxonomic and phylogenetic position. Plant infection tests revealed that all strains were able to form nodules on their original host plant, but with variable nodulation ability and symbiotic effectiveness. All strains were slow-growing, alkali-producing rhizobia in YEMA medium. According to phenotypic test results, the strains were grouped in four main clusters in a UPGMA dendrogram, while they were discriminated into 12 distinct genotypes by 16S rDNA-RFLP. Phylogenetic analyses of 16S rRNA genes and two concatenated housekeeping genes, glnII and recA, distributed the representative strains from each RFLP genotypes into two major clades within Bradyrhizobium genus. Some of these strains were closely related to B. retamae and B. japonicum whereas others represented four novel genospecies. Based on analysis of the symbiotic gene sequence (nodC), all strains were placed in the symbiovar retamae cluster except for SjBA12 which possibly constitutes a new symbiovar within the genus Bradyrhizobium.

Phylogenetic diversity and cross-inoculation of indigenous isolated Bradyrhizobium from nodules of peanut in Liaoning province of China.

Arachis hypogaea. L is a legume of economic importance, which is nodulated by Bradyrhizobium, a slow-growing bacteria. However there is no well characterization of this rhizobia in many areas of China. In the present study, cross-inoculation experiments were performed in cowpea and soybean. The isolated bacteria strains were characterized physiologyically, biochemically and identified through 16S rDNA sequence analysis showing that it belongs to Bradyrhizobium japonicum. The genetic diversity of the seventeen isolated strains were assessed through PCR-RFLP of 16S rDNA and 16S-23S rDNA IGS region. Cross inoculation test indicated that isolates could nodulate cowpea but not soybean. The cluster analysis based on physiological and biochemical characteristics showed the lower correlation between isolates and sites. The isolates were grouped into four clusters based on 16S rDNA gene sequence analysis. Thirteen polymorphisms were variable across all observations in 16S rDNA RFLP and six different IGS types from isolates. The results implies that there was some association between geographical factor and phylogenetic diversity of indigenous Bradyrhizobium isolates.

A ‘Candidatus Phytoplasma omanense’-related strain detected in yellowing grapevine, stunted bindweed and Cixiidae planthoppers in Lebanon

Grapevine yellows (GY) affecting Lebanese vineyards are reported to be associated with ‘Candidatus Phytoplasma solani’, the bois noir (BN) etiological agent. However, during a field survey in June 2014 for BN in Mansoura municipality of West Bekaa, Lebanon, ‘Candidatus Phytoplasma omanense’ was detected in a grapevine sample, cultivar Syrah, exhibiting leaf scorch and discoloration, using a phytoplasma universal nested-PCR and sequencing of the 1.2 kbp 16SrDNA amplicon. The same 1.2 kbp 16SrDNA sequence could be amplified from Hyalesthes obsoletus and Reptalus sp. Cixiidae planthoppers collected on Convolvulus arvensis (bindweed) plants in June 2014 in Aammiq municipality of West Bekaa. Yellowing and stunted bindweed plants collected in 2015 in Kefraya and Aamiq municipalities of West Bekaa were also found infected with the same phytoplasma strain. A 16S rDNA RFLP assay was designed to differentiate this phytoplasma from ‘Ca. P. solani’ and ‘Ca. P. phoenicium’, which are endemic to the Bekaa region. ‘Ca. P. omanense’ is reported for the first time in grapevine and in Cixiidae planthoppers already known to respectively host and vector ‘Ca. P. solani’ strains associated with BN disease in grapevine. This result highlights the need for a precise survey of phytoplasmas associated to grapevine yellows in the Eastern Mediterranean basin, and for investigating the possible role of ‘Ca. P. omanense’ as a new threat to Euro-Mediterranean viticulture.

Molecular diversity of stress-tolerant PGPR rhizobia nodulating clusterbean (Cyamopsis tetragonoloba L.) grown in hyper-arid zone of Rajasthan

A total of 81 rhizobia were retrieved from nodules of clusterbean grown in hyper-arid zone of Rajasthan. Twenty one rhizobial isolates showed combined drought tolerance of 40% concentration of polyethylene glycol 6000 and temperature tolerance at 45°C. All the stress-tolerant rhizobia were authenticated by plant infectivity test and further showed the presence of nitrogen fixation nifH gene. Most of the stress-tolerant rhizobia harbour multiple PGPR traits. The molecular diversity among stress-tolerant rhizobia was accomplished through RFLP of 16S rDNA using restriction enzymes MspI and HaeIII. Dendrogram data showed that all 21 isolates were distributed into two major clusters. Total of 20 genotypes were formed but 13 biotypes were constituted at 80% level of similarity. Out of these, biotype 10 was found to be the most prevalent biotype of hyper-arid zone. Moreover, isolates from same nodule were not 100% similar. It indicated that vast diversity was present among stress-tolerant clusterbean rhizobial isolates.

African origin of Bradyrhizobium populations nodulating Bambara groundnut (Vigna subterranea L. Verdc) in Ghanaian and South African soils.

Flavonoids secreted by legumes play a major role as signal molecules for attracting compatible rhizobia. The aim of this study was to assess and understand the diversity of microsymbionts nodulating Bambara groundnut (Vigna subterranea L. Verdc.) landraces of different seedcoat colours using restriction fragment length polymorphism and phylogenetic analysis. Seedcoat pigmentation of landraces had effect on the diversity of microsymbionts of Bambara groundnut. Even when planted together in one hole, nodulating bradyrhizobia clustered differently. For example, 16S rDNA-RFLP typing of rhizobial samples TUTVSBLM.I, TUTVSCRM.I and TUTVSRDM.I originating respectively from Black, Cream and Red landraces that were co-planted in the same hole at Manga in the Sudano-sahelian savanna, as well as TUTVSCRK.I and TUTVSRDK.I respectively from Cream and Red landraces co-planted at Kpalisogu in the Guinea savanna, revealed different 16S rDNA- RFLP types. Phylogenetic analysis of 16S rDNA, glnII, recA and atpD sequences showed that Vigna subterranea was nodulated specifically by a diverse group of Bradyrhizobium species (e.g. Bradyrhizobium vignae, and a novel group of Bradyrhizobium spp.) in soils from Ghana and South Africa. The recA gene phylogeny showed incongruency with the other housekeeping genes, indicating the possibility of lateral gene transfer and/or recombination events. The grouping of isolates according to symbiotic gene (nifH and nodD) phylogenies revealed inter- and intra-specific symbiotic plasmid transfer and different evolutionary history. The results also showed that a cropping history and physico-chemical environment of soils increased bradyrhizobial diversity in Ghana and South Africa.

Isolation and determination of antimicrobial resistance of Arcobacter species isolated from animal faeces in the Diyarbakir region of Turkey using the 16S rDNA-RFLP method

In this study, the presence of Arcobacter spp. was investigated in the faeces of cattle, sheep, goats, dogs and cloacal swab samples of chickens using the 16S rDNA-RFLP method. The prevalence of Arcobacter in these species was found to be 13%, 12%, 16%, 4% and 33%, respectively. On the other hand, Arcobacter spp. could not be isolated from rabbit faeces. A total of 78 (13%) Arcobacter spp. isolates were obtained from the 500 faecal samples and 100 cloacal swab samples examined in this study. From these 78 Arcobacter isolates, 24 (30.8%), 20 (25.6%), 11 (14.1%), 8 (10.7%), 4 (5.1%), 3 (3.9%) and 2 (2.6%) were identified by 16S rDNA-RLFP as A. cryaerophilus, A. butz- leri, A. skirrowii, A. cloacae, A. cibarius, A. halophilus, and A. nitrofigilis, respectively. All A. cryaerophilus (n = 24) isolates were found to be resistant to cloxacillin; all A. butzleri (n = 20) and A. skirrowii isolates were found to be resistant to penicillin/novobiocin, cefoperazone, tetracycline and cloxacillin. It was determined in this study that clinically healthy cattle, sheep, goats, dogs and chickens are reservoirs of Arcobacter spp.

Microsymbiont diversity and phylogeny of native bradyrhizobia associated with soybean (Glycine max L. Merr.) nodulation in South African soils

The genetic diversity and identification of slow- and fast-growing soybean root nodule bacterial isolates from different agro-climatic regions in Mpumalanga, Limpopo and Gauteng Provinces of South Africa were evaluated. The 16S-rDNA-RFLP analysis of 100 rhizobial isolates and eight reference type strains placed the isolates into six major clusters, and revealed their site-dependent genomic diversity. Sequence analysis of single and concatenated housekeeping genes (atpD, glnII and gyrB), as well as the symbiotic gene nifH captured a considerably higher level of genetic diversity and indicated the dominance of Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum in Mpumalanga, Limpopo and Gauteng Provinces. Gene sequence similarities of isolates with type strains of Bradyrhizobium ranged from 97.3 to 100% for the 16S rDNA, and 83.4 to 100% for the housekeeping genes. The glnII gene phylogeny showed discordance with the other genes, suggesting lateral gene transfer or recombination events. Concatenated gene sequence analysis showed that most of the isolates did not align with known type strains and might represent new species from South Africa. This underscores the high genetic variability associated with soybean Bradyrhizobium in South African soils, and the presence of an important reservoir of novel soybean-nodulating bradyrhizobia in the country. In this study, the grouping of isolates was influenced by site origin, with Group I isolates originating from Limpopo Province and Groups II and III from Mpumlanga Province in the 16S rDNA-RFLP analysis.

FIRST REPORT OF ‘CANDIDATUS PHYTOPLASMA SOLANI’ IN PEPPER AND CELERY IN BOSNIA AND HERZEGOVINA

In August 2015, in Bijeljina (Semberija province of Bosnia and Herzegovina) pepper and celery plants were surveyed for phytoplasma infections. During the survey pepper plants (Capsicum annuum ) of cvs Fortesa, Niska Sipka and Amanda showing stunting and leaf yellowing were observed, while celery plants (Apium graveolens L.) variety ‘Giant Prague’, expressed leaf whitening and stunting; the percentages of symptomatic plants ranged from 20 to 30%, respectively. Leaf samples from symptomatic and asymptomatic plants were tested to verify phytoplasma presence using nested-PCR/RFLP analyses. Results of 16S rDNA RFLP analyses with Tru1I and tuf typing using HpaII (Langer and Maixner, 2004) indicated the presence of a ‘Candidatus Phytoplasma solani’-related strain in all the symptomatic samples tested. There was no variability in the tuf gene and only tuf-type b was detected. Four symptomatic pepper and two celery samples were selected for further characterization amplifying vmp1 and stamp genes (Fialova et al., 2009; Fabre et al., 2011). The nucleotide sequences of the obtained amplicons were submitted to GenBank (accession Nos. KU340846-51 and KU295501-06 for vmp1 and stamp sequences, respectively). Homology with ‘Ca. P. solani’ sequences from database was 96- 100% for the vmp1 and 99-100% for the stamp gene. Combined RFLP analyses using Tru1I and Hpy188I on stamp and RsaI on vmp1 genes distinguished four ‘Ca. P. solani’ lineages indicating the presence of genetic variability among the phytoplasmas studied. This is the first report of ‘Ca. P. solani’ presence in symptomatic pepper and celery in Bosnia and Herzegovina.

16S rDNA sequence analysis of witches’ broom phytoplasma isolates from Chinese jujube in North China

Phytoplasmas are agriculturally important pathogens for more than one thousand plant species. Jujube witches’ broom (JWB), a plant disease caused by phytoplasma, is highly fatal in Chinese jujube, an important fruit tree and medicinal crop in Asia. The 16S rDNA of 15 JWB isolates from 4 provinces in North China were amplified, cloned and sequenced. The isolates were over 99.8 % homologous, indicating that JWB phytoplasma is highly conserved. Phylogenetic analysis of the 16S rDNA sequences from those isolates, together with the representative phytoplasma sequences from other host species, showed that all JWB isolates belong to a subgroup of 16Sr V group and are closely related to Elm yellows (EY) phytoplasma. Virtual RFLP and single-nucleotide polymorphism (SNP) analysis of 16S rDNA also revealed that JWB isolates have a relatively low level of genetic diversity and that their SNP sites exhibit a strong bias toward A/G variation when compared with other phytoplasmas. The high similarity among JWB isolates from different regions and cultivars is consistent with the conserved symptoms of the disease and the wide infectivity of JWB phytoplasmas. This result provides valuable information to better understand the genetic diversity of JWB phytoplasmas.

Paenibacillus herberti sp. nov., an endophyte isolated from Herbertus sendtneri.

Strain R33(T), an endophyte recovered from Herbertus sendtneri, was identified as representing a novel species of the genus Paenibacillus by using a polyphasic taxonomic approach. The novel strain was observed to be a Gram-stain positive, aerobic, rod-shaped, motile and endospore-forming bacterium. The major polar lipids of strain R33(T) were identified as diphosphatidylglycerol, phosphatidylethanolamine, along with lesser amounts of phosphatidylglycerol, three unidentified aminophospholipids, two unidentified phospholipids and two unidentified lipids. The predominant isoprenoid quinone was identified as MK-7. The major fatty acids (>8.0 %) were found to be anteiso-C15:0 (40.0 %), C16:1 ω11c (9.4 %), C16:1 ω7c alcohol (8.5 %) and C16:0 (8.2 %). The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The G+C content of genomic DNA was determined to be 56.9 mol%. The 16S rRNA gene sequence similarities of strain R33(T) to other Paenibacillus species ranged from 91.6 to 97.2 %, with high similarities to Paenibacillus humicus PC-147(T) and Paenibacillus pasadenensis SAFN-007(T). The phylogenetic analyses based on 16S rRNA gene sequences and the partial rpoB gene confirmed that strain R33(T) belongs to the genus Paenibacillus. However, strain R33(T) shows differential molecular characteristics compared to other related Paenibacillus species based on 16S rDNA-RFLP analyses; the DNA-DNA relatedness values between strain R33(T) and P. humicus PC-147(T), and that between strain R33(T) and P. pasadenensis SAFN-007(T), were 35.0 ± 2.0 and 41.4 ± 0.9 %, respectively. Based on its phenotypic, chemotaxonomic and phylogenetic properties, strain R33(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus herberti is proposed (type strain R33(T) = CGMCC 1.15042(T) = DSM 29849(T)).

Detection of the first QnrS gene positivity in aquatic Aeromonas spp. isolates in Turkey

Aeromonas spp. are oxidase positive, gram-negative, facultative anaerobic bacilli that are widely distributed in aquatic environments. A.hydrophila, A.sobria and A.bestiarum may cause severe infections in both human and cold-blooded animals. Environmental persistance of quinolones that are widely used in both human and veterinary medicine plays an important role in the selection of resistant mutants. Plasmid-mediated resistance is one of the main mechanisms involved in quinolone resistance, and qnr, qepA, aac(6')-Ib-cr, oqxAB genes are identified as resistance determinants. Determination of various types of qnr gene in different bacteria mainly in Enterobacteriaceae, suggests that they are widely distributed in nature. Recently, plasmid-mediated quinolone resistance was defined among Aeromonas species isolated from water. The aim of this study was to investigate the presence of qnr genes among aquatic Aeromonas spp. in Turkey. A total of 45 Aeromonas strains isolated from water and fishes collected from three different geographical regions (Aegean, Mediterranean and Blacksea) in Turkey, were included in the study. The isolates were identified at species level by the use of 16S rDNA-RFLP (Restriction fragment length polymorphism) analysis and multiplex polymerase chain reaction (M-PCR). Among the isolates, 20 were identified as A.sobria, 10 as A.hydrophila, nine as A.salmonicida, four as A.bestiarum and two as A.veronii. The plasmid-mediated quinolone resistance determinants, qnrA, qnrB, qnrC and qnrS genes, were investigated by M-PCR, and sequence analysis was performed for nine qnr-positive isolates. According to the sequence analysis of the genes, qnr genes were characterized in six A.sobria, in two A.bestiarum and in one A.hydrophila isolate (9/45; 20%). When the sequence was compared with GenBank database, this gene was found as qnrS2. All qnrS-positive Aeromonas spp. isolates were ciprofloxacin-susceptible, while five of them were resistant to nalidixic acid. This study is the first research about the plasmid-mediated quinolone resistance and the presence of qnrS2 genes among Aeromonas spp. isolated from fishes and water in Turkey. In conclusion, various resistance genes of aquatic bacteria may constitute a potential risk for the transmission of those genes to other bacteria as well as clinical isolates.

Incidence, distribution, economic importance of alfalfa witches’ broom disease in Sistan-Baluchestan (Iran) and characterization of associated phytoplasmas

Alfalfa witches’ broom (AWB) is a devastating disease distributed mainly in tropical provinces of Iran and widespread all over the Sistan-Baluchestan province. The highest incidence was found in Rask (Sarbaz) (70%), Sefidaba (Zabol) (46.26%), Sarboog (Nikshahr) (45.4%), Mirjaveh (Zahedan) (40%), Shams Abad (Saravan) (32.2%), Namardian (Iranshahr) (27%), Kahir (Chabahar) (25.7%) and Najaf Abad (Khash) (17.8%). Dodder inoculation of periwinkle plants with AWBagents from different areas induced undistinguishable symptoms. Detection and identification of AWB associated phytoplasmas was based on characteristic disease symptoms and direct and nested polymerase chain reaction using P1/P7, R16mF2/R16mR2 and R16F2n/R16R2 primer pairs. RFLP and phylogenetic analyses of 16S rDNA showed that AWB associated phytoplasmas from Sistan-Baluchestan belong to peanut witches’ broom (16SrII) group. Virtual digestion of R16F2n/R16R2 sequence of a Nikshahr AWB strain exhibited RFLP profiles referable to those of members of the 16SrII phytoplasma group, subgroup D.

Arcobacter ebronensis sp. nov. and Arcobacter aquimarinus sp. nov., two new species isolated from marine environment

Two strains recovered from mussels (F128-2T) and sea water (W63T) were characterized as Arcobacter sp., but they could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR) and rpoB gene analysis. The 16S rRNA gene sequence similarity to the type strains of all Arcobacter species ranged from 92.2% to 96.7% with strain F128-2T, and from 94.1% to 99.4% with strain W63T, the most similar being A. bivalviorum (CECT 7835T) and A. defluvii (CECT 7697T), respectively. The phylogenetic analyses of 16S rRNA, and the concatenated sequences of gyrB, gyrA, rpoB, atpA and hsp60 genes confirmed that strains F128-2T and W63T belonged to two new lineages within the genus Arcobacter. Moreover, both strains showed differential phenotypic characteristics and MALDI-TOF mass spectra from all other Arcobacter species. Therefore, it has been demonstrated the existence of two new Arcobacter species and the proposed names are Arcobacter ebronensis (type strain F128-2T=CECT 8441T=LMG 27922T), and Arcobacter aquimarinus (type strain W63T=CECT 8442T=LMG 27923T).

First Report of a 16SrII-C Phytoplasma Associated with Asymptomatic Acid Lime (Citrus aurantifolia) in Brazil.

At present, the principal bacterial disease of citrus in Brazil is Huanglongbing, caused by the alpha-proteobacterium 'Candidatus Liberibacter spp.' (although a phytoplasma of the 16SrIX group is also associated with this disease [4]). While there is a wide diversity of phytoplasmas in crop species in Brazil (3), there have been no reports of symptoms associated with phytoplasma in Brazilian citrus. Asymptomatic infections of citrus cannot be excluded as a possibility and such plants could serve as a reservoir of phytoplasma inoculum. The aim of this study was to assess the presence of phytoplasma in asymptomatic Citrus aurantifolia (acid lime) in Brazil. Thirty-three leaf samples (young leaves from the upper canopies) were randomly collected from different plants in the states of Minas Gerais (n = 23), Santa Catarina (n = 2), and São Paulo (n = 8). Two additional samples of C. limonia ('Rangpur' lime) and one of C. latifolia ('Persian' or 'Tahiti' lime) were collected in Minas Gerais. Total DNA extraction was performed using NucleoSpin Plant II Kit (Macherey-Nagel) according to the manufacturer's recommendations. PCR was carried out with a universal P1/P7 primer set followed by nested primers R16F2n/R16R2 (2). Additionally, direct PCR was performed using primers specific for phytoplasma immune-dominant membrane protein IMP3F/IMP3R (1). 'Rangpur' and 'Tahiti' lime were not infected with phytoplasma. Of the C. aurantifolia samples, 52% were positive for phytoplasma in the direct and nested PCR assays. The numbers of positive samples in Minas Gerais, Santa Catarina, and São Paulo states were 12, 1, and 4, respectively. Of these, five were selected for DNA purification and 1,246-bp fragments were ligated to the pGEM T-easy vector (Promega) and partial 16Sr DNA was sequenced. Nucleotide sequences of Brazilian phytoplasma strains BR:MG:FNS10:2011, BR:MG:FNS53:2011, BR:SP:FNS73:2011, BR:SC:FNS86:2011, and BR:MG:FNS126:2012 (GenBank Accession Nos. KJ158173, KJ158174, KJ158175, KJ158176, and KJ158177, respectively) were subjected to RFLP analyses. The 16S rDNA RFLP in silico patterns for the five strains were identical to each other and to Cactus witches'-broom phytoplasma (16SrII-C subgroup, AJ293216). In addition, the highest similarity coefficient (5) and nucleotide sequence identity of Brazilian phytoplasma strains were 0.99 and 99%, respectively, with Cactus witches'-broom phytoplasma. PCR-RFLP analyses using the enzymes Bstu I, EcoR I, and Hpa II were consistent with RFLP in silico results, showing the same pattern as the 16SrII-C subgroup. Phylogenetic analyses based on 16S rDNA sequences (1,246 bp) demonstrated that all the Brazilian strains grouped in the same clade with other representative sequences from the 16S rDNAII group. To confirm the absence of any macroscopic symptoms, morphological characteristics of 10 uninfected and 10 phytoplasma-infected plants randomly selected from a single field in Minas Gerais were analyzed. There were no significant differences in leaf area, stalk diameter, or numbers of leaves, flowers, or fruits per branch. To our knowledge, this is the first report of the 16SrII-C subgroup phytoplasma associated with C. aurantifolia in Brazil, and the first report of asymptomatic citrus plants infected with phytoplasma.

English

Rhizobacteria are possible alternative to chemical fertilizers and used to manage phyto-pathogens. In this study, rhizobacteria isolated from rice rhizosphere of coastal agriculture system of South Andaman, India were evaluated in vitro for their plant growth promoting property (PGP), antagonistic activity and genetic diversity using PCR-RFLP. From 63 isolates, 35 were positive for salt tolerance of upto 0.5 M NaCl and 27 isolates upto 1.0 M NaCl. The biochemical characterization showed that most of the isolates were positive for one or other plant growth promoting property. Antagonistic activity against the fungi Sclerotium rolfsii revealed that 13 isolates have significantly inhibited mycelial growth of S. rolfsii as compared to the control but the isolate HMI 4, HMI 5, HMI 10, HMI 14, HMI 16 and HMI 17 were most efficient in percentage inhibition of test pathogen, caused more than 35% inhibition. RFLP of 16s rDNA showed homogenous population distributed randomly at 59% similarity. 16s rDNA sequencing result showed that most predominant population were Pseudomonas spp. followed by Enterobacter and Bacillus spp. Five best isolates were evaluated in vitro for PGP activity in rice and were found promising. The present study has resulted in the isolation of PGP and antagonistic Pseudomonas (HMI 16, HMI 17) and Bacillus spp. (HMI 14) that could be useful to design strategies to use these isolates as inoculants in sustainable agriculture of Andaman, India. Key words: Andaman, Bacillus, phosphate solubilization, PCR-RFLP, siderophore.

Prevalence and antimicrobial susceptibility of Arcobacter species in cow milk, water buffalo milk and fresh village cheese

In this study, the presence of Arcobacter spp. was examined in cow milk (n=50), water buffalo (WB) milk (n=50) and fresh village cheese (n=50) samples. The 16S rDNA-RFLP method was used for the identification of Arcobacter spp. The disc diffusion method was used to investigate the susceptibility of all strains identified to 18 different antimicrobial substances. The most commonly isolated Arcobacter species were found to be Arcobacter butzleri (38.89%), Arcobacter cryaerophilus (22.23%) and Arcobacter skirrowii (11.12%) in cow milk; A. cryaerophilus (33.33%), Arcobacter cibarius (20.83%) and A. butzleri (12.50%) in WB milk; and A. skirrowii (28.57%), A. butzleri (21.43%) and A. cryaerophilus (14.29%) in fresh village cheese. This is the first study to identify the presence of Arcobacter nitrofigilis, Arcobacter cloacae, Arcobacter halophilus, Arcobacter bivalviorum and A. cibarius species in analyzed samples. It was found that all of the A. cryaerophilus (n:16) isolates were resistant to cefoperazone, cloxacillin and penicillin G; all of the A. skirrowii (n:12) and A. butzleri (n:10) isolates were resistant to cefoperazone, tetracycline, ampicillin, erythromycin, cloxacillin and penicillin G. It was concluded that cow milk, WB milk and fresh village cheese samples are an important source of Arcobacter species and pose a risk to public health.

Re-identification of Aeromonas isolates from rainbow trout and incidence of class 1 integron and β-lactamase genes

Forty-eight Aeromonas isolates from rainbow trout previously identified by the 16S rDNA-RFLP technique were re-identified using 2 housekeeping genes (gyrB and rpoD). After sequencing the prevalences of the species were A. veronii (29.2%), A. bestiarum (20.8%), A. hydrophila (16.7%), A. sobria (10.4%), A. media (8.3%), A. popoffii (6.2%), A. allosaccharophila (2.1%), A. caviae (2.1%), A. salmonicida (2.1%) and one isolate (2.1%) belongs to a candidate new species “Aeromonas lusitana”. Coincident identification results to the 16S rDNA-RFLP technique were only obtained for 68.8% of the isolates. PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC-PCR) indicated that the 48 isolates belonged to 33 different ERIC genotypes. Several genotypes were isolated from different farms and organs in the same fish, indicating a systemic dissemination of the bacteria. The presence of genes (blaIMP, blaCphA/IMIS, blaTEM, blaSHV and intI1) that encode extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs) and class 1 integrons were studied by PCR. Only 39.6% (19/48) of the strains showed the presence of one or more resistance genes. The gene blaCphA/IMIS was detected in 29.2% of the isolates, followed by the intI1 (6.2%) and blaSHV (4.2%) genes. The variable region of class 1 integrons of the 3 positive isolates was sequenced revealing the presence of the gene cassette aadA1 (aminoglycoside transferase) that plays a role in streptomycin/spectinomycin resistance.

Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4–6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA–RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP–16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.