Rhizosphere-associated Alcaligenes and Bacillus strains that induce resistance against blast and sheath blight diseases, enhance plant growth and improve mineral content in rice.

AIMS: To examine the biocontrol activities of five rhizobacterial strains (i.e. Alcaligenes faecalis strains Bk1 and P1, Bacillus amyloliquefaciens strain Bk7 and Brevibacillus laterosporus stains B4 and S5), to control the rice blast and sheath blight diseases in greenhouse and to study their possible modes of action. METHODS AND RESULTS: Five potential plant growth-promoting rhizobacterial (PGPR) strains isolated from rice rhizospheres were tested for in vitro antifungal activities against Magnaporthe oryzae, Rhizoctonia solani, Botrytis cinerea and Fusarium graminearum. In vitro trials showed that three strains, Bk1, P1 and Bk7, were able to unanimously suppress the mycelial growth of the target pathogens. In greenhouse, the application of these three PGPR strains significantly suppressed the incidences of rice blast and sheath blight diseases. At 2 weeks after pathogen inoculation, the highest percentages of disease suppression were noted for Alc. faecalis strain Bk1 (72%) for rice blast, Alc. faecalis strain P1 (71%) for sheath blight, followed by B. amyloliquefaciens strain Bk7. Moreover, these strains significantly improved the plant growth, enriched the content of mineral nutrients in seedlings and increased the expression of major defence-related rice genes. All three strains were marked positive for phosphate solubilization, the production of indoleacetic acid, ammonia and siderophores and catalase activity. In addition, these strains were able to form biofilms and carried multiple lipopeptide biosynthetic genes as revealed by multiplex PCR. CONCLUSION: This study reports new potential biocontrol agents for blast and sheath blight diseases of rice. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to better understanding of the mechanisms involved in interaction between beneficial rhizobacteria, fungal pathogens and host plants.

Two EST-derived marker systems for cultivar identification in tree peony.

Tree peony (Paeonia suffruticosa Andrews), a woody deciduous shrub, belongs to the section Moutan DC. in the genus of Paeonia of the Paeoniaceae family. To increase the efficiency of breeding, two EST-derived marker systems were developed based on a tree peony expressed sequence tag (EST) database. Using target region amplification polymorphism (TRAP), 19 of 39 primer pairs showed good amplification for 56 accessions with amplicons ranging from 120 to 3,000 bp long, among which 99.3% were polymorphic. In contrast, 7 of 21 primer pairs demonstrated adequate amplification with clear bands for simple sequence repeats (SSRs) developed from ESTs, and a total of 33 alleles were found in 56 accessions. The similarity matrices generated by TRAP and EST-SSR markers were compared, and the Mantel test (r = 0.57778, P = 0.0020) showed a moderate correlation between the two types of molecular markers. TRAP markers were suitable for DNA fingerprinting and EST-SSR markers were more appropriate for discriminating synonyms (the same cultivars with different names due to limited information exchanged among different geographic areas). The two sets of EST-derived markers will be used further for genetic linkage map construction and quantitative trait locus detection in tree peony.

Haplotype diversity at the Pi-ta locus in cultivated rice and its wild relatives.

The Pi-ta gene in rice confers resistance to races of Magnaporthe oryzae that contain AVR-Pita. Pi-ta encodes a predicted cytoplasmic receptor protein with a nucleotide-binding site and a leucine-rich domain. A panel of 51 Oryza accessions of AA genome species Oryza sativa, O. glaberrima, O. rufipogon, O. nivara, and O. barthii, and CC genome species O. officinalis were sequenced to investigate the diversity present in the exon and intron regions of the Pi-ta gene. Two major clades were identified, consisting of 16 different sequences with numerous insertion and deletions. Only one Pi-ta resistance allele was identified despite DNA sequences revealing 16 Pi-ta variants. Most differences were identified in the intron region, and obvious selection of any motif was not observed in the coding region of Pi-ta variants. Reverse-transcription polymerase chain reaction analysis of seedlings revealed that all Pi-ta variants were expressed with or without pathogen inoculation. The 15 Pi-ta variants can be translated into nine proteins highly similar to the Pi-ta protein. Resistance to M. oryzae expressing AVR-Pita correlates with alanine and susceptibility correlates with serine at position 918 of Pi-ta in most accessions examined. These data confirm that a single amino acid controlling resistance specificity underlies the evolution of resistance of Pi-ta genes in rice.

Gene actions of QTLs affecting several agronomic traits resolved in a recombinant inbred rice population and two backcross populations.

To understand the types of gene action controlling seven quantitative traits in rice, we carried out quantitative trait locus (QTL) mapping in order to distinguish between the main-effect QTLs (M-QTLs) and digenic epistatic QTLs (E-QTLs) responsible for the trait performance of 254 recombinant inbred lines (RILs) from rice varieties Lemont/Teqing and two backcross hybrid (BCF1) populations derived from these RILs. We identified 44 M-QTL and 95 E-QTL pairs in the RI and BCF1 populations as having significant effects on the mean values and mid-parental heterosis of heading date, plant height, flag leaf length, flag leaf width, panicle length, spikelet number and spikelet fertility. The E-QTLs detected collectively explained a larger portion of the total phenotypic variation than the M-QTLs in both the RI and BCF1 populations. In both BCF1 populations, over-dominant (or under-dominant) loci were more important than additive and complete or partially dominant loci for M-QTLs and E-QTL pairs, thereby supporting prior findings that overdominance resulting from epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice.

Genetic transformation of Leymus chinensis with the PAT gene through microprojectile bombardment to improve resistance to the herbicide Basta.

Chinese leymus [Leymus chinensis (Trin.) Tzvel.] is a perennial grass (tribe Gramineae) that is widely distributed throughout northern China and Mongolia where it is produced as a forage product. Severe production losses due to weed growth have serious economic consequences, and as non-selective herbicides not only kill the weeds but are also harmful to this forage grass, the introduction of a foreign gene for resistance to the herbicide Basta is necessary since this species lacks herbicide resistance. We have investigated the transformation of a gene for phosphinothricin acetyltransferase (PAT) through microprojectile bombardment in Chinese leymus. Calli from immature inflorescences cultured on N6 medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/l of glutamine were bombarded. The bombarded calli survived on selection medium with 1.0 mg/l of phosphinothricin (PPT). Twenty-three plantlets regenerated from resistant calli on differentiation medium supplemented with 1.0 mg/l 6-benzylaminopurine, 1.0 mg/l kinetin, and 1.0 mg/l PPT, and five of these regenerated plantlets survived on rooting medium with 1.0 mg/l of PPT. PCR and Southern blotting analyses indicated that the PAT gene had been integrated into the genomes of two Chinese leymus plantlets and that the gene was stably transferred to its clonal offsprings. There were no other phenotypic effects associated with transgene expression during vegetative growth except tolerance to the herbicide Basta.

An effective method for axillary bud culture and RAPD analysis of cloned plants in tetraploid black locust.

An efficient micropropagation technique by axillary bud multiplication was established for cloning tetraploid black locust tree (Robinia pseudoacacia L.). The result showed that the optimal medium for shoot multiplication and elongation was Murashige and Skoog (MS) medium supplemented with 0.5 mg/l 6-benzylaminopurine in combination with 0.5 mg/l kinetin and 0.1 mg/l 1-naphthaleneacetic acid. The best medium for rooting was half-strength MS medium with 0.25 mg/l indole-3-butyric acid. In the present report, we examined the genetic fidelity of the micropropagated plants by the randomly amplified polymorphic DNA (RAPD) method with 25 primers. The cloned plants of tetraploid black locust showed complete stability.

[Genetic analysis of cry1Ab gene of Bt rice].

Improved histochemical staining for GUS activity, PCR and Western blotting were used to analyse the progeny population of Bt rice crossed with conventional rice varieties. A total of 392 plants expressing Bt toxin protein were found in 394 GUS positive plants. The result demonstrated that cry1Ab gene closely inherited and expressed with reporter gene gus. GUS assays indicated that cry1Ab gene of Bt rice inherits as a single dorminant gene. Southern hybridization confirmed cry1Ab gene can inherit stabily in the progenies of Bt rice. Mendelian segregation of reporter gene gus was also observed in F2, BC1 and BC1F2 progenies, which indicated that cry1Ab gene inherits as a single dorminant gene in the progenies of Bt rice crossed with conventional rice varieties.

Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. I. Biomass and grain yield.

To understand the genetic basis of inbreeding depression and heterosis in rice, main-effect and epistatic QTL associated with inbreeding depression and heterosis for grain yield and biomass in five related rice mapping populations were investigated using a complete RFLP linkage map of 182 markers, replicated phenotyping experiments, and the mixed model approach. The mapping populations included 254 F(10) recombinant inbred lines derived from a cross between Lemont (japonica) and Teqing (indica) and two BC and two testcross hybrid populations derived from crosses between the RILs and their parents plus two testers (Zhong 413 and IR64). For both BY and GY, there was significant inbreeding depression detected in the RI population and a high level of heterosis in each of the BC and testcross hybrid populations. The mean performance of the BC or testcross hybrids was largely determined by their heterosis measurements. The hybrid breakdown (part of inbreeding depression) values of individual RILs were negatively associated with the heterosis measurements of their BC or testcross hybrids, indicating the partial genetic overlap of genes causing hybrid breakdown and heterosis in rice. A large number of epistatic QTL pairs and a few main-effect QTL were identified, which were responsible for >65% of the phenotypic variation of BY and GY in each of the populations with the former explaining a much greater portion of the variation. Two conclusions concerning the loci associated with inbreeding depression and heterosis in rice were reached from our results. First, most QTL associated with inbreeding depression and heterosis in rice appeared to be involved in epistasis. Second, most ( approximately 90%) QTL contributing to heterosis appeared to be overdominant. These observations tend to implicate epistasis and overdominance, rather than dominance, as the major genetic basis of heterosis in rice. The implications of our results in rice evolution and improvement are discussed.

Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. II. Grain yield components.

The genetic basis underlying inbreeding depression and heterosis for three grain yield components of rice was investigated in five interrelated mapping populations using a complete RFLP linkage map, replicated phenotyping, and the mixed model approach. The populations included 254 F(10) recombinant inbred lines (RILs) derived from a cross between Lemont (japonica) and Teqing (indica), two backcross (BC) and two testcross populations derived from crosses between the RILs and the parents plus two testers (Zhong413 and IR64). For the yield components, the RILs showed significant inbreeding depression and hybrid breakdown, and the BC and testcross populations showed high levels of heterosis. The average performance of the BC or testcross hybrids was largely determined by heterosis. The inbreeding depression values of individual RILs were negatively associated with the heterosis measurements of the BC or testcross hybrids. We identified many epistatic QTL pairs and a few main-effect QTL responsible for >65% of the phenotypic variation of the yield components in each of the populations. Most epistasis occurred between complementary loci, suggesting that grain yield components were associated more with multilocus genotypes than with specific alleles at individual loci. Overdominance was also an important property of most loci associated with heterosis, particularly for panicles per plant and grains per panicle. Two independent groups of genes appeared to affect grain weight: one showing primarily nonadditive gene action explained 62.1% of the heterotic variation of the trait, and the other exhibiting only additive gene action accounted for 28.1% of the total trait variation of the F(1) mean values. We found no evidence suggesting that pseudo-overdominance from the repulsive linkage of completely or partially dominant QTL for yield components resulted in the overdominant QTL for grain yield. Pronounced overdominance resulting from epistasis expressed by multilocus genotypes appeared to explain the long-standing dilemma of how inbreeding depression could arise from overdominant genes.

Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers.

Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China. Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season. In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads. Under natural infestation by the C. suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts. Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively. Damage to the control plants from artificial infestation by the S. incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged. The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.