rDNA Sequence Similarity Research Articles

Fluorescent pseudomonads (FPs) as a potential biocontrol and plant growth promoting agent associated with tomato rhizosphere

A total of 87 FPs have been isolated from tomato (Lycopersicon esculentum) rhizosphere and evaluated by dual plate assay method for the antimicrobial activity of Ralstonia solanacearum. Of which, 30 FPs showed antagonistic activity against R. solanacearum with the zone of inhibitions (ZOI) ranging from 19 mm to 28 mm. Similarly, antagonistic FPs significantly controlled various fungal phytopathogens such as Rhizoctonia solani (6.3 mm−20 mm), Macrophomina phaseolina (8.7 mm−19.3 mm), Fusarium oxysporum (7.3 mm−30.7 mm), and Sclerotium rolfsii (5.3 mm−21 mm). The cell-free culture filtrate of Pseudomonas sp VSMKU3054 significantly suppressed the fungal pathogens and increased the root and shoot length of the tomato seedlings compared to the control. Furthermore, the antagonistic FPs effectively produced lytic enzymes and antimicrobial traits such as amylase (6), protease (30), cellulase (28), pectinase (12), chitinase (22), gelatinase (27), siderophore (28), hydrogen cyanide (25), phosphate solubilization (30), and Indole acetic acid (IAA) (23). Genetic diversity of FPs was assessed by BOX-PCR with specific primers revealed that two distinct clusters were observed, whereas, RFLP analyses were showed that 5 to 16 bands ranged from 75 bp to 1.2 bp with two major clusters using restriction enzyme HaeIII. The antibiotic encoding genes were detected from FPs, of which 10 FPs were positive for 2,4-diacetylphloroglucinol (DAPG), 9 FPs were positive for pyoleuterin (PLT), 10 FPs were positive for hydrogen cyanide (HCN) and none of them obtained pyrrolnitrin (PRN) with respective primers. Based on the superior antagonistic isolates against R. solanacearum and phytofungal pathogens and other antimicrobial traits, the isolate VSMKU3054 was selected for further studies. Based on the morphological, physiological and 98% of 16 s rDNA sequence similarity, the selected isolate VSMKU3054 was identified as Pseudomonas fluorescens. Further, the potential isolate P. fluorescens VSMKU3054 and cell free culture filtrate remarkably enhance tomato seedling growth such as root and shoot length, fresh and dry weight and vigor index compared to control. This study indicated that rhizospheric isolates of FPs have more potential for plant growth promotion and plant protection from plant pathogenic microbes.

Identification of Gangesia oligonchis Roitman & Freze, 1964 (Cestoda: Onchoproteocephalidea) from Tachysurus fulvidraco Richardson in central China: implications for the validity of Gangesia pseudobagrae Chen, 1962.

Owing to the brief and incomplete original description of Gangesia pseudobagrae Chen, 1962 (Cestoda: Onchoproteocephalidea) and high morphological similarity to Gangesia oligonchis Roitman & Freze, 1964 parasitising the same host Tachysurus fulvidraco Richardson, the taxonomic validity of G. pseudobagrae in China remains questionable. Therefore, we sampled and identified specimens of Gangesia Woodland, 1924 from the intestine of T. fulvidraco from three lakes in central China. Morphologically, the sampled specimens almost perfectly corresponded both to G. oligonchis and the limited available description of G. pseudobagrae: rostellum-like organ armed with a single complete circle of hooks (24-31 in number); four uniloculate suckers covered with minute hooklets; genital pore irregularly alternated; testes medullary, spherical to oval; ovary medullary, bi-lobed, follicular; cirrus-sac thick-walled and long; uterus medullary. 28S rDNA sequence also exhibited the highest similarity to G. oligonchis (99.4-99.7%). Phylogenetic analysis showed that 11 individuals of Gangesia from the three lakes in China clustered with G. oligonchis from Russia (no sequence of G. pseudobagrae available on GenBank). Based upon the high similarity of morphology and high similarity of 28S rDNA sequences, the specimens of Gangesia from T. fulvidraco in central China were identified as G. oligonchis. Our results indicate that there is only one species of Gangesia in T. fulvidraco from the Palaearctic region, and thereby support the proposed synonymisation of G. pseudobagrae and G. oligonchis.

First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan.

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan.

Setomelanomma holmii (Pleosporales, Phaeosphaeriaceae) on living spruce twigs in Europe and North America

Setomelanomma holmii M. Morelet, previously known only from the type specimen in France, was discovered in the U.S.A. (Kansas and Wisconsin) and Canada (Ontario) on living twigs of spruce (Picea pungens and Picea glauca). This fungus was grown from ascospores and compared with the ex-holotype culture. Morphology and ITS rDNA sequence similarities indicate that S. holmii belongs in the Pleosporales, Phaeosphaeriaceae. Sequence analysis of the SSU nrDNA places S. holmii in a clade containing members of the Leptosphaeriaceae and Phaeosphaeriaceae. Setomelanomma holmii is redescribed and illustrated based on the holotype and North American specimens.Key words: Loculoascomycetes, Phaeosphaeriaceae, Picea, Pleosporales, needle chlorosis.

ASCRIPTION OF Nt-ISOLATES OF RHIZOCTONIA SOLANI TO ANASTOMOSIS GROUP 2-1 (AG-2-1) ON ACCOUNT OF rDNA-ITS SEQUENCE SIMILARITY

A homogeneous group of Rhizoctonia solani isolates from tobacco, which showed intermediate features between anastomosis groups BI and 2-1 (AG-BI and AG- 2-1), was recently characterized. It was not conclusively assigned to any previously known AG and was provisionally named ‘Nt-isolates’. The rDNA sequence similarity in the ITS1 region of the Nt-isolates was evaluated and a high similarity (> 90%) was found with AG-2- 1 tester isolates, while it was remarkably lower with tester isolates from AG-BI and other AG-2 subgroups. Therefore, despite a low anastomosis frequency, Nt-isolates are to be considered members of AG-2-1.

Relationships among Colletotrichum isolates from fruit-rots assessed using rDNA sequences

Isolates of Colletotrichum associated with fruit rots in New Zealand were used to test the applicability of morphological, cultural, and rDNA sequence data to clarify relationships within this genus. Morphological and cultural features were used to recognize 16 distinct groups among the New Zealand fruit-rotting isolates. These included the currently accepted species C. coccodes, C. musae, C. orbiculare, and Glomerella miyabeana as well as 3 C. acutatum like groups and 9 C. gloeosporioides-like groups. Analysis of rDNA sequences from the D2 region allowed the isolates to be separated into 4 broad groups (the relationships of C. coccodes remaining unresolved), the various morphological/cultural groups clustering as subgroups among the rDNA sequence groups. The same 4 groups were recognized when rDNA sequences from the New Zealand isolates were compared with those from Colletotrichum isolates from other hosts and other countries. Taxonomic treatment of the molecular and morphological/cultural data is discussed. A trinomial group-species concept may provide the best approach to subgeneric classification within Colletotrichum. The species name (based on rDNA sequence similarity and supporting morphological and cultural features) indicates the broad group within Colletotrichum to which the isolate belongs; the subspecific name provides additional information on host specialisation, morphological variation, etc. However, before the utility of this concept can be properly assessed, the evolutionary and genetic relationships between the various morphological and sequence-defined groups need to be better understood. If such a scheme is shown to be practical in a biological sense then a number of taxonomic and nomenclatural problems will need to be resolved before it could be adopted.

Relationships among Colletotrichum Isolates from Fruit-Rots Assessed Using rDNA Sequences

Isolates of Colletotrichum associated with fruit rots in New Zealand were used to test the applicability of morphological, cultural, and rDNA sequence data to clarify relationships within this genus. Morphological and cultural features were used to recognize 16 distinct groups among the New Zealand fruit-rotting isolates. These included the currently accepted species C. coccodes, C. musae, C. orbiculare, and Glomerella miyabeana as well as 3 C. acutatum like groups and 9 C. gloeosporioides-like groups. Analysis of rDNA sequences from the D2 region allowed the isolates to be separated into 4 broad groups (the relationships of C. coccodes remaining unresolved), the various morphological/cultural groups clustering as subgroups among the rDNA sequence groups. The same 4 groups were recognized when rDNA sequences from the New Zealand isolates were compared with those from Colletotrichum isolates from other hosts and other countries. Taxonomic treatment of the molecular and morphological/cultural data is discussed. A trinomial group-species concept may provide the best approach to subgeneric classification within Colletotrichum. The species name (based on rDNA sequence similarity and supporting morphological and cultural features) indicates the broad group within Colletotrichum to which the isolate belongs; the subspecific name provides additional information on host specialisation, morphological variation, etc. However, before the utility of this concept can be properly assessed, the evolutionary and genetic relationships between the various morphological and sequence-defined groups need to be better understood. If such a scheme is shown to be practical in a biological sense then a number of taxonomic and nomenclatural problems will need to be resolved before it could be adopted.