Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. ‘Azul’ in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1α). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, β-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.     

Variability of Indian Isolates of Tilletia indica Assessed by Pathogenicity and Molecular Markers

Twenty isolates of Tilletia indica collected from sites in North and North‐western India showed pathogenic variation on 18 host differentials. Sixteen aggressive pathotypes were identified on the basis of percent coefficient of infection (PCI). Two major clusters were apparent in the dendrogram; cluster 1 comprised 13 isolates and cluster two consisted of seven isolates. One of the isolate Kashipur had a high PCI on most of the host differentials compared to other isolates. Polymerase chain reaction‐based random amplified polymorphic DNA (PCR – RAPD) analysis also divided isolates into two major clusters, one comprising of 5 isolates collected from hill and foot‐hill sites and another group comprising of 15 isolates collected from plain sites. Thus, the clusters identified based on PCI did not match closely with those identified by molecular analysis based on RAPD. Although diversity among the isolates of T. indica was absent in the rDNA‐ITS region, our study based on pathogenicity and molecular markers confirms the existence of great diversity in the pathogen, also shifting of ‘hot spot’ areas from one place to another within Karnal bunt prevailing areas.     

First record and preliminary evaluation of Mucor hiemalis as biocontrol agent on inflorescence brown rot incidence of date palm

In Egypt, inflorescence brown rot disease of date plam trees caused by Thielaviopsis paradoxa De Syenes causing high losses of pollen grains and fruits yield productivity. Infection occurs early on spathes even when it still hidden in the leaf bases. White mycelium of pathogenic fungi grows on inflorescence then turned to brown when fungus spores abundant. Isolation trails from diseased spathes showed brown rot, yielded three genus of fungal i.e. Aspergillus niger (25%), Mucor hiemalis (25%) and T. paradoxa (50%). Pathogenicity test by using fungal isolates and male inflorescence of data indicate that, all isolates of T. paradoxa were able to induce brown rot of inflorescence. Isolates of T. paradoxa were differed in pathogenic activity for producing inflorescence brown rot symptom. Also, A. niger isolate could cause slightly decay on inflorescence. Meanwhile, all isolates of M. hiemalis recorded as non pathogenic. In vitro, dual culture technique by using M. hiemalis showed antagonistic properties against T. paradoxa. Scanning electron microscopy (SEM) study revealed that, pollen grains of date palm are susceptible to infection by T. paradoxa, accompanied by complete lyses and ruptured. SEM examination of inflorescence treated by each of M. hiemalis or/and T. paradoxa showed that M. hiemalis was able to colonisation on inflorescence and reduced colonisation of T. paradoxa on inflorescence and pollen grains. Preliminary evaluation of M. hiemalis as a biocontrol agent showed that, spray of inflorescence with M. hiemalis suspension two days before or after infestation by T. paradoxa were reduced brown rot of inflorescence than the control. Spraying of spathes by M. hiemalis before infestation by pathogen was highly effective in reduction brown rot incidence compared with spraying after infestation. These results help to explain the role of M. hiemalis in the suppression and biological control of T. paradoxa.     

A contribution to characterisation of genetic variation in some natural Polish populations of Elymus repens (L.) Gould and Elymus hispidus (Opiz) Melderis (Poaceae) as revealed by RAPD markers*

To determine the relative importance of clonal growth and sexual reproduction, the Randomly Amplified Polymorphic DNA (RAPD) method was used to study genetic diversity and clonal structure of six populations of Elymus repens and four populations of Elymus hispidus from Poland. These outbreeding species are virtually self‐sterile and form widely spreading and long‐lived rhizomes. Using 12 primers, a total of 150 unambiguous RAPD fragments were amplified and scored. Results of AMOVA showed no significant genetic distinction between morphologically distinguished varieties of E. repens and E. hispidus. E. repens had slightly higher intra‐specific genetic polymorphism than E. hispidus; the percentage of polymorphic bands per population ranged from 38 to 49 and from 19 to 38 respectively. Clonal diversity measured using the Simpson diversity index (D) indicated different contributions of clonal reproduction in particular populations of E. repens (D: 0.20–0.72). Populations of E. hispidus were dominated by one or a few clones, which were generally restricted to a single population (D: 0.00–0.22). RAPD revealed that most genetic diversity resided within populations of the two studied species, suggesting that, despite their clonal character, propagation by seeds contributes considerably to reproduction of E. repens and E. hispidus.     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Genetic and virulence variation in Fusarium oxysporum f. sp. cepae causing root and basal rot of common onion in Iran

Root and basal rot of common onion (Allium cepae L.) caused by Fusarium oxysporum f. sp. cepae is one of the most important diseases causing tremendous losses in onion‐growing areas worldwide. In this study, random amplified polymorphic DNA (RAPD), intersimple sequence repeats (ISSR) and virulence studies were conducted to analyse 26 F. oxysporum f. sp. cepae isolates obtained from the main onion‐growing regions of Iran, including Fars, Azerbaijan and Isfahan states. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion; however, virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

In vitro Antagonistic Interactions Between Endophytic Basidiomycetes of Oil Palm (Elaeis guineensis) and Ganoderma boninense

Ganoderma boninense is a white rot basidiomycete that causes basal stem rot disease of oil palm (Elaeis guineensis). The aims of this study were to identify endophytic basidiomycetes occurring naturally within oil palm and to assess their potential as biocontrol agents against G. boninense strain PER71 in vitro. In total, 376 isolates were recovered from samples collected from the root, stem and leaves of oil palm using Ganoderma‐selective medium. Ten of these isolates (2.7% of the total 376 isolates) were identified as basidiomycetes on the basis of clamp connections and the production of poroid basidiomes after incubation in glass jars containing PDA medium for 7–12 days. The isolates were identified using ITS rDNA sequencing as Neonothopanus nambi (five isolates), Schizophyllum commune (four isolates) and Ganoderma orbiforme (one isolate). The N. nambi isolates showed the greatest antagonistic activity against G. boninense, based on 73–85% inhibition of the radial growth measurements of G. boninense in dual culture and 76–100% inhibition of G. boninense growth in a culture filtrate assay. Possible modes of action for the antagonism shown by N. nambi against G. boninense in vitro include competition for substrate availability, space and the production of non‐volatile metabolites or antibiotics that inhibited the growth of G. boninense. Further in vivo investigations are required to determine the ability of N. nambi isolates to colonize oil palm seedlings and to protect oil palm from infection when challenged with G. boninense.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.     

AFLP and RAPD Characterization of Phaeoacremonium aleophilum Associated with Vitis vinifera Decline in Spain

The genetic diversity among Spanish isolates of the fungus Phaeoacremonium aleophilum, one of the major causes of grapevine decline, was determined using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) techniques. Using RAPD, a large genetic variation was observed among 36 Pm. aleophilum single‐spore cultures, with 76 (82.6%) polymorphic bands generated by 12 RAPD primers. A neighbour‐joining dendrogram showing the RAPD patterns of diversity revealed four groups of haplotypes. The Bayesian and principal components clustering analysis revealed three groups of haplotypes. When more than one isolate of Pm. aleophilum was obtained from a single vine, different haplotypes were found. Seventeen single‐spore isolates were used for AFLP analysis. Five primer combinations produced 358 scorable markers, of which 309 (86.3%) were polymorphic. The analysis based on genetic distance as well as clustering analysis confirmed three main groups largely in agreement with those returned by the RAPD results. The Mantel correlation between the RAPD and AFLP distance matrices ranged from R = 0.5931 to R = 0.6294. The high level of haplotype diversity among the RAPD and AFLP markers suggests that sexual reproduction and genetic recombination may occur between Pm. aleophilum haplotypes in Spain. The AFLP approach revealed a greater number of polymorphic markers. A relationship between the genetic profile of the infecting isolate of Pm. aleophilum and the age or decline symptoms of the grapevines may exist.     

Effects of clonality on the genetic variability of rare,insular species: the case of Ruta microcarpa from the Canary Islands

Many plant species combine sexual and clonal reproduction. Clonal propagation has ecological costs mainly related to inbreeding depression and pollen discounting; at the same time, species able to reproduce clonally have ecological and evolutionary advantages being able to persist when conditions are not favorable for sexual reproduction. The presence of clonality has profound consequences on the genetic structure of populations, especially when it represents the predominant reproductive strategy in a population. Theoretical studies suggest that high rate of clonal propagation should increase the effective number of alleles and heterozygosity in a population, while an opposite effect is expected on genetic differentiation among populations and on genotypic diversity. In this study, we ask how clonal propagation affects the genetic diversity of rare insular species, which are often characterized by low levels of genetic diversity, hence at risk of extinction. We used eight polymorphic microsatellite markers to study the genetic structure of the critically endangered insular endemic Ruta microcarpa. We found that clonality appears to positively affect the genetic diversity of R. microcarpa by increasing allelic diversity, polymorphism, and heterozygosity. Moreover, clonal propagation seems to be a more successful reproductive strategy in small, isolated population subjected to environmental stress. Our results suggest that clonal propagation may benefit rare species. However, the advantage of clonal growth may be only short‐lived for prolonged clonal growth could ultimately lead to monoclonal populations. Some degree of sexual reproduction may be needed in a predominantly clonal species to ensure long‐term viability.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers

Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson's diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Reproductive modes of three Ligularia weeds (Asteraceae) in grasslands in Qinghai-Tibet Plateau and their implications for grassland management

Ligularia virgaurea, L. sagitta and L. przewalskii are noxious weeds that are widely distributed in psychro-grasslands in the east of Qinghai-Tibet Plateau of China. However, little is known about reproductive modes and genetic diversity in the populations of L. virgaurea and L. sagitta under different environmental conditions, although such information is available for L. przewalskii. In this study, two experiments were conducted to examine the ex situ clonal growth of these weeds, and to determine the effects of reproductive modes on the diversity of their populations, using random amplified polymorphic DNA (RAPD) analysis. Transplanted seedlings of L. sagitta and L. virgaurea showed considerable clonal growth by forming rhizomes and ramets. The RAPD analysis showed that all individuals collected from natural populations were genetically different, indicating that no individuals originated from clonal growth. It is concluded that L. virgaurea and L. sagitta have the capacity for clonal growth, but the expression of their clonality is constrained by their growing conditions in natural grasslands. Sexual reproduction plays the major role in their population diversity in natural habitats, which requires that the control measures for these weeds should be targeted at the sexually reproducing populations.     

Molecular identification of isolates of <Emphasis Type="Italic">Peronosclerospora sorghi</Emphasis> from maize using PCR-based SCAR marker

The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.