RAPD Marker Linked to a Gene Conferring Resistance to Race IB-9 of Pyricularia grisea in a Somaclone of the Rice Cultivar Araguaia

The gene Pi-ar confers resistance to Pyricularia grisea in a somaclone of the upland rice cultivar Araguaia developed from callus culture of immature panicles. The somaclone SC09 exhibited resistant reaction to all of the 182 P. grisea test isolates belonging to 15 different races. The study on inheritance showed that the resistance to pathotype IB-9 of P. grisea is monogenic and dominant. In order to identify marker linked to this gene, the F₂ population from a cross between the highly susceptible cultivar Lijiangxintuanheigu (LTH) and the somaclone SC09 of rice cultivar Araguaia was screened using RAPD primers. Initially, the polymorphism between parents, the cultivar LTH and somaclone SC09 was analyzed using 577 random 10-bp primers. The susceptible and resistant bulks of the F₂ population, along with DNA of the two parents were tested with 176 primers that differentiated susceptible and resistant parents. Thirty-six primers differentiated the susceptible and resistant bulks, as well as the cultivar LTH of the somaclone SC09. However, one primer OPK17 was found to be closely linked (5.3 cM) to the resistance gene of somaclone and this can be used in the marker assisted selection.     

Isolation of TC/AG Repeat Microsatellite Sequences for Fingerprinting Rice Blast Fungus and Their Possible Horizontal Transfer to Plant Species

Genome fingerprinting has been a major role in characterization of population structure and analysis of the variability in phytopathogenic fungi. In order to characterize Korean rice blast fungal isolates, the genomic DNAs were digested with Alu I endonuclease and subsequent PCR amplifications using random decamer primers with combinations of microsatellite primers had been carried out. This Alu-Inter SSR technique revealed high polymorphism among the Korean blast fungal isolates. Then, fragments from the Alu-Inter SSR analysis were isolated to be used as probes in Southern hybridization, which also revealed high polymorphism between isolates to distinguish individuals. The sequences of the isolated fragments contained TC/AG tandem repeats interspersed with a 30 bp direct repeat. In gel blot analysis, the isolated TC/AG repeat microsatellite sequences were proved to be useful for characterizing the isolates in blast fungi in addition to the conventional MGR (Magnaporthe grisea repeat) probes. One interesting point was that the rice blast fungus derived TC/AG repeat microsatellite sequences were abundant in non-rice blast fungi and plant species, but not in other fungi and yeasts. A discussion on the possible horizontal gene transfer between phytopathogenic fungi and host plants is presented.     

Purification and anti-fungal activity of chitinase against Pyricularia grisea in finger millet

Pseudomonas fluorescens isolate 1 (Pfl) protected finger millet plants treated with the ragi blast fungus, Pyricularia grisea, by upto 27% depending on the cultivar. Induction of pathogenesis-related proteins, viz., chitinase by Pfl isolate, was studied against Py. grisea. The activity of chitinase from plants treated with Pfl was significantly higher than the control plant after pathogen inoculation in all cultivars tested. Chitinase in the cultivars, with and without challenge by Py. grisea, revealed changes in the isoform pattern by western blot analysis. Chitinase was purified by affinity chromatography from the Pfl-treated leaves. It showed a single band at 57 kDa after SDS-PAGE. Western blot analysis using barley chitinase antiserum confirmed a 57 kDa chitinase. The chitinase had anti-fungal activity against Py. grisea in vitro. This revised version was published online in August 2006 with corrections to the Cover Date.     

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.     

Mapping of a Magnaporthe grisea locus affecting rice (Oryza sativa) cultivar specificity

Magnaporthe grisea causes rice blast, the most important fungal disease of rice. The segregation of genes controlling virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in this host-parasite interaction. Full-sib progeny and parent isolates Guy11 and 2539 of M. grisea were inoculated onto rice (Oryza sativa) cultivar CO39 and five near-isogenic lines (NILs) of CO39. Each NIL contained a different single gene affecting resistance to specific isolates of M. grisea. No differential interactions between NILs and progeny or parents were observed; parents and progeny pathogenic on CO39 were pathogenic on all five NILs. Segregation ratios of 101 full-sib progeny, 117 progeny from full-sib parents, and 109 backcross progeny, indicated a common single gene affecting pathogenicity on CO39 and the five NILs. A subset of the above 327 isolates (43 fullsib progeny, 37 progeny from full-sib parents, and 32 backcross progeny) were inoculated onto rice cultivar 51583; all were pathogenic, indicating that cultivar specificity to CO39 was segregating in this population of isolates. The locus controlling cultivar specificity, named avrCO39, was mapped to chromosome 1 using a subset of the progeny previously used to construct an RFLP map of M. grisea. The closest reported RFLP markers were 11.8 (estimated 260 kb) and 17.2 cM (estimated 380 kb) away and provide starting points on either side of the locus for a chromosome walk to clone the locus.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Mating-type Distribution and Fertility Status in Magnaporthe grisea Populations from Argentina

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.     

Reaction of incompatible isolate of Pyricularia grisea on rice leaves stripped of epicuticular wax reflects blast conduciveness of soils

Upland rice cultivars were evaluated in the greenhouse for susceptibility to the rice blast disease caused by Pyricularia grisea Sacc., on two upland soils from the Philippines previously considered to be “blast conducive” and “blast non-conducive”. Under monocyclic inoculation tests plants grown in conducive soil showed significantly greater lesion development than plants of the same cultivar grown in non-conducive soil: cultivars considered to be susceptible to the isolates used showed increased number of susceptible-type lesions; resistant cultivars showed increased number of hypersensitive resistant-type lesions. A similar effect was observed under polycyclic tests where several generations of the pathogen were allowed to develop on the test plants. Dilution of conducive soil with non-conducive soil resulted in a corresponding reduction of disease severity, although this was most pronounced on resistant cultivars. Removal of leaf epicuticular waxes (LEW) using organic solvents increased the number of resistant-type lesions on resistant cultivars grown in both soils following inoculation. Susceptible plants were not suitable for quantifying the relative blast conduciveness of a soil because of the extreme environmental sensitivity of the bioassay and the tendency of lesions to coalesce. Comparing numbers of resistant-type lesions on leaves of plants stripped of LEW and inoculated with an incompatible P. grisea isolate among plants grown in different soils proved to be a satisfactory means of distinguishing the relative blast conduciveness of soils under controlled conditions. This method was field tested in eastern India and results corroborated farmer assessment of which soils were blast conducive. Using incompatible isolate-cultivar combinations and LEW-free leaves is proposed as a simple bioassay for assessing blast conduciveness of soils and should prove useful in regional characterization of rice blast risk.     

RAPD analysis of Fusarium Isolates Causing “Mango Malformation” Disease in Pakistan

Mango Malformation (MM) disease is a major constraint to mango production. A total of 20 Fusarium isolates from MM-affected mango plants were collected from 14 locations in Pakistan and assessed for genetic diversity using the random amplified polymorphic DNA (RAPD) technique. A total of 393 fragments were amplified after screening with 50 random primers. The amplifications with 45 primers identified scoreable polymorphisms among the isolates. A genetic similarity matrix based on Nei and Li’s index determined coefficients ranging from 46.46% to 92.51%. These coefficients were used to construct a dendrogram using the UPGMA algorithm. The isolates grouped into two main clusters, comprising 13 and 7 isolates respectively, at a genetic relatedness of 52%. Within the clusters, Fusarium isolates were not necessarily related either by geographic origin or by the mango cultivar from which they were isolated. RAPD proved a reproducible and tractable means of differentiating Fusarium isolates. These findings also suggest that some infections originate not from adjacent plants within an orchard but from geographically distant areas; indicating that most probably infection occurs in nurseries prior to plants being transported around the country for subsequent cultivation, and that improved plant hygiene could significantly curb MM infection and spread.     

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F₁ progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi–asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The χ² test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR–Pit and AVR–Pia, were identified. Genetic linkage analysis showed that the SSR marker m355–356 is closely linked to AVR–Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR–Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.     

Genetic Structure of Pyricularia grisea (Cooke) Sacc. Isolates from Italian Paddy Fields

Rice blast disease, caused by the fungus Pyricularia grisea (Cooke) Sacc., is responsible for considerable damages in rice crops in Italy and in other parts of the world. This study was conducted in order to investigate the genetic structure of a P. grisea population in the Po area, the largest rice area in Italy. Rice leaves showing blast symptoms were collected in three successive years (1998–2000) and 43 P. grisea monoconidial culture samples were isolated from infected rice leaves. Fungal DNAs were obtained from mycelia. Moreover, six additional P. grisea DNA samples representative for the five characterized European lineages were also investigated. All 49 DNAs were fingerprinted using the Pot2‐based repetitive polymerase chain reaction specific for the blast pathogen. Unweighted pair‐group method with arithmetic averages cluster analysis shows the presence of three main Italian lineages. Within lineages, similarity was higher than 80%. Samples representative of the three of five known European lineages grouped within these three Italian lineages confirming the presence of three European lineages in Italy. Furthermore, cluster analysis shows the presence of two new haplotypes never found before in the Italian lineage.     

Didymocarpus geitleri,a remarkable new species ofGesneriaceae with deceptive pollen flowers

Didymocarpus geitleri spec. nov. is described from Sungai Pandan, Kuantan distr., Pahang, Peninsular Malaysia. Distinctive features include: leaden-grey leaves with long, erect hairs on the upper side and whitish, campanulate flowers with a long exserted, bright yellow style. The colouring of the latter is caused by densely arranged glands. Together with two yellow blotches in front of the filament insertion, the style apparently acts as an anther (pollen) dummy.The paper and the species is dedicated to my greatly respected teacher of Systematic Botany, Prof. DrLothar Geitler, on the occasion of his 90th birthday.     

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.     

PGPR mediates induction of pathogenesis-related (PR) proteins against the infection of blast pathogen in resistant and susceptible ragi [Eleusine coracana (L.) Gaertner] cultivars

The Pseudomonas fluorescens isolate 1 (Pf1) was found to protect the ragi [Eleusine coracana (L.) Gaertner] blast fungus, Pyricularia grisea. Induction of defense proteins viz. chitinase, β-1,3 glucanase, peroxidase (PO) and polyphenol oxidase (PPO) by the Pf1 isolate was studied against P. grisea. Chitinase in a resistant, susceptible and commonly used cultivar with and without challenge inoculation of P. grisea, revealed changes in the isoform pattern by UV illumination after staining the gel with fluorescent brightner 28. Native PAGE (polyacrylamide gel electrophoresis) of PO showed the single isoform in all the treatments including the control and a significant increase in the intensity of the band in the inoculated control and Pf1 treatment in all the varieties. Isoform analysis of PPO showed the induction of PPO in P. fluorescens treated plants challenged with P. grisea.     

Nitrogen form and silicon nutrition effects on resistance to blast disease of rice

The effect of N form and Si nutrition on rice (Oryza sativa L.) susceptibility to blast disease (caused by Pyricularia oryzae Cav.) was assessed in the greenhouse with nutrient solution culture. The N form supplied to the susceptible cultivar IR50 affected the relative infection efficiency (RIE) of P. oryzae measured as lesions/cm² leaf. Plants given NO₃ ^- were more susceptible than plants receiving NH₄ ⁺-N. This result may partially explain why plants grown in nonflooded soil, where NO₃ ^- is the main source of inorganic N, are more susceptible to blast than plants grown in flooded soils, where NH₄ ⁺ is the main inorganic N source. Nitrate-N and Mn concentration were higher in leaf blades of plants grown with NO₃ ^-. Total-N, Si, and Fe concentration were not affected by N form. The addition of Si significantly increased IR50 resistance to blast. With 2.2 mol m^-3 Si in solution, RIE values were lower by more than 90% than the control with no Si added in solution. The effect of Si accumulation in leaves at various positions was further studied in cultivars having differing levels of resistance (IR50, IR36, and IAC165). Silicon addition significantly reduced RIE in the three cultivars. Silicon concentration in the topmost leaves (the only leaves showing typical blast lesions) was not significantly different among the three cultivars when 2.2 mol m^-3 Si was used. Silicon was an important component in the mechanism of resistance to blast and it was effective regardless of the original level of resistance of the cultivar used. Contribution from the Agronomy Unit, Agronomy-Physiology-Agroecology Division, International Rice Research Institute (IRRI), P.O. Box 933, 1099 Manila, Philippines, and Colegio de Postgraduados, Mexico. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree.     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

Transgenic indica rice expressing Mirabilis jalapa antimicrobial protein (Mj-AMP2) shows enhanced resistance to the rice blast fungus Magnaporthe oryzae

Mj-AMP2, a knottin-type antimicrobial peptide, in vitro inhibits the growth of several plant pathogenic fungi including Magnaporthe oryzae. We demonstrate that transgenic rice (Oryza sativa L.) plants expressing the Mj-AMP2 gene show enhanced resistance to M. grisea, the causal agent of the rice blast disease. Mj-AMP2 was efficiently expressed and the level of Mj-AMP2 ranged from 0.32% to 0.38% of the total protein in the transgenic rice plants. In vitro inhibitory activity assays with the crude protein extract from transgenic rice indicated that the Mj-AMP2 protein produced was biologically active. Constitutive expression of Mj-AMP2 in transgenic rice reduces the growth of M. grisea by 63% with respect to untransformed control plant, and no effect on plant phenotype was observed. Transgene expression of Mj-AMP2 gene was not accompanied by an induction of pathogenesis-related (PR) gene expression indicating that the transgene product itself is directly active against the pathogen. The results presented in this study suggest that the Mj-AMP2 gene could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Somaclonal variants resistant to sugarcane mosaic virus and their agronomic characterization

Summary The sugarcane mosaic virus is a pathogen that causes severe disease to sugarcane. New varieties resistant to insects and pathogens have been developed in the last 70 yr through sugarcane breeding programs, but this takes between 10 and 15 yr. Tissue culture techniques are used as an aid for sugarcane improvement to increase desirable agronomic characteristics, such as disease resistance. In the present work, we report the generation of somaclonal variants from sugarcane (Saccharum spp.) cultivar PR62258 susceptible to Sugarcane Mosaic Virus, by somatic embryogenesis. These new variants identified as AT626 and BT627 are resistant to Sugarcanes Mosaic Virus strains A and B, respectively. We established an indirect enzyme linked inmunosorbent assay (ELISA) to test the presence of the viral particles in plants, and its was demonstrated that the leaves of resistant somaclones do not contain viral particles. The field performance of the somaclones AT626 and BT627 was similar to the field performance of the mother plant PR62258.     

Quantitative trait variation in phenotypically normal regenerants of cotton

Summary Somaclonal variation for quantitative traits could affect the practical utilization of regenerants in cotton improvement. Three groups of experimental lines were derived to analyze variation, including one control group from the explant-source cultivar and two groups of R₃ somaclones from different R₀’s (R₀ = initial regenerant) free of observable chromosomal rearrangements. A three-environment field trial was conducted to evaluate group means, genetic variance, and line performance. Mean seedcotton yields of the somaclonal groups were reduced by 21 and 26% relative to the Coker 310 standard at two locations, but lint percentage and certain fiber properties were improved. Group-by-environment interactions were significant (P<0.05) for 10 of the 12 measured traits. Genetic variance tended to decrease in the somaclones, plant height being an exception. Line performance of the somaclones indicated that 50-boll weight, seed index, and fiber length did not reach the Coker 310 group means. These data suggest that genetic gain will be improved if regenerants of cotton are self-pollinated and the progenies evaluated for quantitative traits before crossing somaclones with the explantsource cultivar or other elite germplasm.