<Emphasis Type="Italic">GNS4</Emphasis>, a novel allele of <Emphasis Type="Italic">DWARF11</Emphasis>, regulates grain number and grain size in a high-yield rice variety

Background

Rice plays an extremely important role in food safety because it feeds more than half of the world’s population. Rice grain yield depends on biomass and the harvest index. An important strategy to break through the rice grain yield ceiling is to increase the biological yield. Therefore, genes associated with organ size are important targets for rice breeding.

Results

We characterized a rice mutant gns4 (grain number and size on chromosome 4) with reduced organ size, fewer grains per panicle, and smaller grains compared with those of WT. Map-based cloning indicated that the GNS4 gene, encoding a cytochrome P450 protein, is a novel allele of DWARF11 (D11). A single nucleotide polymorphism (deletion) in the promoter region of GNS4 reduced its expression level in the mutant, leading to reduced grain number and smaller grains. Morphological and cellular analyses suggested that GNS4 positively regulates grain size by promoting cell elongation. Overexpression of GNS4 significantly increased organ size, 1000-grain weight, and panicle size, and subsequently enhanced grain yields in both the Nipponbare and Wuyunjing7 (a high-yielding cultivar) backgrounds. These results suggest that GNS4 is key target gene with possible applications in rice yield breeding.

Conclusion

GNS4 was identified as a positive regulator of grain number and grain size in rice. Increasing the expression level of this gene in a high-yielding rice variety enhanced grain yield. GNS4 can be targeted in breeding programs to increase yields.

     

A novel Rice QTL <Emphasis Type="Italic">qOPW11</Emphasis> Associated with Panicle Weight Affects Panicle and Plant Architecture

Background

The improvement of rice yield is a crucial global issue, but evaluating yield requires substantial efforts. Rice yield comprises the following indices: panicle number (PN), grain number per panicle (GN), 1000-grain weight, and percentage of ripened grain. To simplify measurements, we analyzed one panicle weight (OPW) as a simplified yield index that integrates GN, grain weight, and percentage of ripened grain, and verified its suitability as a proxy for GN and grain weight in particular.

Results

Quantitative trait locus (QTL) analysis using 190 recombinant inbred lines derived from Koshihikari (large panicle and small grain) and Yamadanishiki (small panicle and large grain), japonica cultivars detected three QTLs on chromosomes 5 (qOPW5), 7 (qOPW7) and 11 (qOPW11). Of these, qOPW5 and qOPW11 were detected over two years. qOPW5 and qOPW7 increased OPW, and qOPW11 decreased it at Yamadanishiki alleles. A chromosome segment substitution line (CSSL) with a genomic segment from Yamadanishiki substituted in the Koshihikari genetic background harboring qOPW5 increased grain weight. qOPW11 had the largest genetic effect of QTLs, which was validated using a CSSL. Substitution mapping using four CSSLs revealed that qOPW11 was located in the range of 1.46 Mb on chromosome 11. The CSSL harboring qOPW11 decreased primary and secondary branch numbers, culm length, and panicle length, and increased PN.

Conclusions

In this study, three QTLs associated with OPW were detected. The CSSL with the novel and largest QTL, qOPW11, differed in some traits associated with both panicle and plant architecture, indicating different functions for the meristem in the vegetative versus the reproductive stages. qOPW5 coincided with an identified QTL for grain width and grain weight, suggesting that qOPW5 was affected by rice grain size. OPW can be considered a useful trait for efficient detection of QTLs associated with rice yield.

     

Effect of water management on soil respiration and <Emphasis Type="Italic">NEE</Emphasis> of paddy fields in Southeast China

Water management is an important factor in regulating soil respiration and the net ecosystem exchange of CO₂ (NEE) between croplands and atmosphere. However, how water management affects soil respiration and the NEE of paddy fields remains unexplored. Thus, a 2-year field experiment was carried out to study the effects of controlled irrigation (CI) during the rice season on the variation of soil respiration and NEE, with flooding irrigation (FI) as the control. A decrease of irrigation water input by 46.39% did not significantly affect rice yield but significantly increased irrigation water use efficiency by 0.99 kg m^?3. The soil respiration rate of CI paddy fields was larger than that of FI paddy fields except during the ripening stage. Natural drying management during the ripening stage resulted in a significant increase of the soil respiration rate of the FI paddy fields. Variations of NEE with different water managements were opposite to soil respiration rates during the whole rice growth stages. Total CO₂ emission of CI paddy fields through soil respiration (total R _soil) increased by 11.66% compared with FI paddy fields. The increase of total R _soil resulted in the significant decrease of total net CO₂ absorption of CI paddy fields by 11.57% compared with FI paddy fields (p < 0.05). There were inter-annual differences of soil respiration and the NEE of paddy fields. Frequent alternate wetting and drying processes in the CI paddy fields were the main factors influencing soil respiration and NEE. CI management slightly enhanced the rice dry matter amount but accelerated the consumption and decomposition of soil organic carbon and significantly increased soil respiration, which led to the decrease of net CO₂ absorption. CI management and organic carbon input technologies should be combined in applications to achieve sustainable use of water and soil resources in paddy fields.     

Dissection and fine-mapping of two QTL for grain size linked in a 460-kb region on chromosome 1 of rice

Background

Grain size is a key determinant of grain weight and a trait having critical influence on grain quality in rice. While increasing evidences are shown for the importance of minor-effect QTL in controlling complex traits, the attention has not been given to grain size until recently. In previous studies, five QTL having small effects for grain size were resolved on the long arm of chromosome 1 using populations derived from indica rice cross Zhenshan 97///Zhenshan 97//Zhenshan 97/Milyang 46. One of them, qTGW1.2c that was located in a 2.1-Mb region, was targeted for fine-mapping in the present study.

Results

Firstly, the qTGW1.2c region was narrowed down into 1.1 Mb by determining genotypes of the cross-over regions using polymorphic markers newly developed. Then, one BC₂F₉ plant that was only heterozygous in the updated QTL region was identified. A total of 12 populations in generations from BC₂F_11:12 to BC₂F_15:16 were derived and used for QTL mapping. Two QTL linked in a 460-kb region were separated. The qGS1-35.2 was delimited into a 57.7-kb region, containing six annotated genes of which five showed nucleotide polymorphisms between the two parental lines. Quantitative real-time PCR detected expression differences between near isogenic lines for qGS1-35.2 at three of the six annotated genes. This QTL affected grain length and width with opposite allelic directions, exhibiting significant effect on ratio of grain length to width but showing little influence on yield traits. The other QTL, qGW1-35.5, was located within a 125.5-kb region and found to primarily control grain width and consequently affect grain weight.

Conclusions

Our work lays a foundation for cloning of two minor QTL for grain size that have potential application in rice breeding. The qGS1-35.2 could be used to modify grain appearance quality without yield penalty because it affects grain shape but hardly influences grain yield, while qGW1-35.5 offers a new gene recourse for enhancing grain yield since it contributes to grain size and grain weight simultaneously.

     

<Emphasis Type="Italic">OsLAP6/OsPKS1</Emphasis>, an orthologue of <Emphasis Type="Italic">Arabidopsis PKSA/LAP6</Emphasis>, is critical for proper pollen exine formation

Background

Male fertility is crucial for rice yield, and the improvement of rice yield requires hybrid production that depends on male sterile lines. Although recent studies have revealed several important genes in male reproductive development, our understanding of the mechanisms of rice pollen development remains unclear.

Results

We identified a rice mutant oslap6 with complete male sterile phenotype caused by defects in pollen exine formation. By using the MutMap method, we found that a single nucleotide polymorphism (SNP) variation located in the second exon of OsLAP6/OsPKS1 was responsible for the mutant phenotype. OsLAP6/OsPKS1 is an orthologous gene of Arabidopsis PKSA/LAP6, which functions in sporopollenin metabolism. Several other loss-of-function mutants of OsLAP6/OsPKS1 generated by the CRISPR/Cas9 genomic editing tool also exhibited the same phenotype of male sterility. Our cellular analysis suggested that OsLAP6/OsPKS1 might regulate pollen exine formation by affecting bacula elongation. Expression examination indicated that OsLAP6/OsPKS1 is specifically expressed in tapetum, and its product is localized to the endoplasmic reticulum (ER). Protein sequence analysis indicated that OsLAP6/OsPKS1 is conserved in land plants.

Conclusions

OsLAP6/OsPKS1 is a critical molecular switch for rice male fertility by participating in a conserved sporopollenin precursor biosynthetic pathway in land plants. Manipulation of OsLAP6/OsPKS1 has potential for application in hybrid rice breeding.

     

Identification and validation of a novel major QTL for harvest index in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Background

Harvest index (HI) in rice is defined as the ratio of grain yield (GY) to biomass (BM). Although it has been demonstrated that HI is significantly related to yield and is considered as one of the most important traits in high-yielding rice breeding, HI-based high-yielding rice breeding is difficult due to its polygenic nature and insufficient knowledge on the genetic basis of HI. Therefore, searching for rice varieties with high HI and mapping genes associated with high HI can facilitate marker-assisted breeding for high HI in rice.

Results

Yuexiangzhan, a popular indica cultivar with good reputation of high HI was crossed with Shengbasimiao, an indica cultivar with lower HI to develop a recombinant inbred line population, and QTL mapping for HI and its component traits was conducted. In total, five QTLs for HI, three QTLs for GY, and six QTLs for BM were detected in two-year experiments. Among the three GY QTLs, one co-located with the HI QTL on chromosome 8, while the other two co-located with the two tightly-linked BM QTLs on chromosome 3. The co-located QTLs in each of the chromosomal regions produced additive effects in the same direction. Particularly, the HI QTL on chromosome 8, qHI-8, could be detected across two years and explained 42.8% and 44.5% of the phenotypic variation, respectively. The existence of qHI-8 was confirmed by the evaluation of the near isogenic lines derived from a residual heterozygous line, and this QTL was delimitated to a 1070 kb interval by substitution mapping.

Conclusion

In the present study, the detected GY QTLs overlapped with both HI QTL and BM QTL, suggesting a positive relationship between GY and HI or BM, respectively. With an understanding of the genetic basis for grain yield, harvest index and biomass, it is possible to achieve higher yield through enhancing HI and BM by pyramiding the favorable alleles for the two traits via marker-assisted selection (MAS). As qHI-8 has a large phenotypic effect on HI and expresses stably in different environments, it provides a promising target for further genetic characterization of HI and MAS of high HI in rice breeding.

     

The Effect of Alfalfa Residue Incorporation on Soil Bacterial Communities and the Quantity of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Microsclerotia in Potato Fields in the Columbia Basin of Washington State,USA

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is one of the most important diseases of potato in North America. Soil incorporation of alfalfa residues prior to planting potato could be a nonchemical Verticillium wilt management tactic by reducing the number of viable microsclerotia in field soil. Verticillium dahliae microsclerotia were quantified in field soils where organic material from alfalfa was incorporated, and numbers of microsclerotia were compared to fields where alfalfa residue was not incorporated. In addition, bacterial metagenomics was utilized to characterize soils where organic material from alfalfa was or was not incorporated to determine if alfalfa residue incorporation facilitates the formation of soils that suppress or kill V. dahliae microsclerotia. The number of V. dahliae microsclerotia in soil was greater (P = 0.0003) in fields where crop residue was incorporated than fields without incorporation when chloropicrin was used as a fumigant. Conversely, the number of V. dahliae microsclerotia observed in potato plants did not differ (P = 0.4020) between fields where residues were or were not incorporated if chloropicrin was used. Alfalfa residue incorporation did not significantly alter the soil bacterial metagenome compared to fields not subject to residue incorporation in both years of study. Despite these conclusions, the method can be employed to analyze the effect of grower practices with the intent of linking a field practice to increasing soil bacterial diversity and decreasing Verticillium wilt severity on potato.     

Agronomic fortification of rice grains with secondary and micronutrients under differing crop management and soil moisture regimes in the north Indian Plains

Although the System of Rice Intensification (SRI) has been reported to produce higher paddy (Oryza sativa L.) yields with better-quality grains, little research has addressed the latter claim. This study investigated the interactive effects of rice cultivation methods with different irrigation schedules and plant density on the uptake and concentration of sulfur (S), zinc (Zn), iron (Fe), manganese (Mn) and copper (Cu) in the grain and straw of two rice cultivars during two rainy seasons in the northern plains of India. As the two seasons differed in amounts of rainfall, there were impacts of soil moisture differences on nutrient uptake. Plots with SRI cultivation methods enhanced the grain uptake and concentrations of S, Zn, Fe, Mn and Cu by 36, 32, 28, 32 and 63%, respectively, compared to conventional transplanting (CT). Under SRI management, the highest concentrations of S, Zn and Cu in the grain and straw occurred with irrigation intervals scheduled at 3 days after disappearance of ponded water (DADPW; 3_D), whereas Fe and Mn concentrations in the grain and straw were higher with irrigation at 1 DADPW (1 _D ) compared with plots under 3 _D or 5 DADPW (5 _D ). The higher nutrient uptakes were also manifested in higher grain yield in 1 _D and 3 _D plots (by 9 and 6%, respectively) compared with 5 _D . Wider spacing (25 × 25 cm) compared with closer spacing (20 × 20 cm) significantly increased yield and the uptake and concentrations of all the said nutrients in the grains. When comparing the performance of two cultivars, the total uptakes of Zn, Fe, Mn and Cu in both grain and straw were significantly more in Hybrid 6444 than the improved variety Pant Dhan 4. Overall, SRI crop management compared to CT practices led to more biological fortification of rice grains with respect to S and the four micronutrients studied, giving a concomitant yield advantage of about 17% on average in this region.     

Updating the elite rice variety Kongyu 131 by improving the <Emphasis Type="Italic">Gn1a</Emphasis> locus

Background

Kongyu 131 is an elite japonica rice variety of Heilongjiang Province, China. It has the characteristics of early maturity, superior quality, high yield, cold tolerance and wide adaptability. However, there is potential to improve the yield of Kongyu 131 because of the relatively few grains per panicle compared with other varieties. Hence, we rebuilt the genome of Kongyu 131 by replacing the GRAIN NUMBER1a (Gn1a) locus with a high-yielding allele from a big panicle indica rice variety, GKBR. High-resolution melting (HRM) analysis was used for single nucleotide polymorphism (SNP) genotyping.

Results

Quantitative trait locus (QTL) analysis of the BC₃F₂ population showed that the introgressed segment carrying the Gn1a allele of GKBR significantly increased the branch number and grain number per panicle. Using 5 SNP markers designed against the sequence within and around Gn1a, the introgressed chromosome segment was shortened to approximately 430 Kb to minimize the linkage drag by screening recombinants in the target region. Genomic components of the new Kongyu 131 were detected using 220 SNP markers evenly distributed across 12 chromosomes, suggesting that the recovery ratio of the recurrent parent genome (RRPG) was 99.89%. Compared with Kongyu 131, the yield per plant of the new Kongyu 131 increased by 8.3% and 11.9% at Changchun and Jiamusi, respectively.

Conclusions

To achieve the high yield potential of Kongyu 131, a minute chromosome fragment carrying the favorable Gn1a allele from the donor parent was introgressed into the genome of Kongyu 131, which resulted in a larger panicle and subsequent yield increase in the new Kongyu 131. These results indicate the feasibility of improving an undesirable trait of an elite variety by replacing only a small chromosome segment carrying a favorable allele.

     

Dissecting combining ability effect in a rice NCII-III population provides insights into heterosis in <Emphasis Type="Italic">indica-japonica</Emphasis> cross

Background

Combining ability is a measure for selecting elite parents that make the highest contributions to hybrid performance. However, the genetic bases of combining ability and how they contributed to heterosis is seldomly known.

Results

We constructed a both NCII and NCIII population derived from an indica-japonica cross to study the relationship among parental performance, combining ability and hybrid performance of 11 agronomic traits. Among them, specific combining ability is more important to grain yield than parental performance and general combining ability. We performed linkage analyses to phenotypic values and combining ability of all 11 traits in Doubled haploid lines and its two backcross populations and identified 108 QTLs in total. Among these QTLs, four known loci, Sd1, Ghd7, Ghd8 and DEP1 contribute a lot to GCA effects of agronomic traits except grain yield and seed setting rate. Three QTLs, Ghd8, S5 and qS12, contribute a lot to SCA effects of grain yield and present overdominace.

Conclusions

Our study provides insights into the genetic bases of combining ability and heterosis and will promote the improvements of indica-japonica hybrid breeding.

     

Genome-wide association study and candidate gene analysis of rice cadmium accumulation in grain in a diverse rice collection

Background

Cadmium (Cd) accumulation in rice followed by transfer to the food chain causes severe health problems in humans. Breeding of low Cd accumulation varieties is one of the most economical ways to solve the problem. However, information on the identity of rice germplasm with low Cd accumulation is limited, particularly in indica, and the genetic basis of Cd accumulation in rice is not well understood.

Results

Screening of 312 diverse rice accessions revealed that the grain Cd concentrations of these rice accessions ranged from 0.12 to 1.23?mg/kg, with 24 accessions less than 0.20?mg/kg. Three of the 24 accessions belong to indica. Japonica accumulated significantly less Cd than indica (p < 0.001), while tropical japonica accumulated significantly less Cd than temperate japonica (p < 0.01). GWAS in all accessions identified 14 QTLs for Cd accumulation, with 7 identified in indica and 7 identified in japonica subpopulations. No common QTL was identified between indica and japonica. The previously identified genes (OsHMA3, OsNRAMP1, and OsNRAMP5) from japonica were colocalized with QTLs identified in japonica instead of indica. Expression analysis of OsNRAMP2, the candidate gene of the novel QTL (qCd3–2) identified in the present study, demonstrated that OsNRAMP2 was mainly induced in the shoots of high Cd accumulation accessions after Cd treatment. Four amino acid differences were found in the open reading frame of OsNRAMP2 between high and low Cd accumulation accessions. The allele from low Cd accumulation accessions significantly increased the Cd sensitivity and accumulation in yeast. Subcellular localization analysis demonstrated OsNRAMP2 expressed in the tonoplast of rice protoplast.

Conclusion

The results suggest that grain Cd concentrations are significantly different among subgroups, with Cd concentrations decreasing from indica to temperate japonica to tropical japonica. However, considerable variations exist within subgroups. The fact that no common QTL was identified between indica and japonica implies that there is a different genetic basis for determining Cd accumulation between indica and japonica, or that some QTLs for Cd accumulation in rice are subspecies-specific. Through further integrated analysis, it is speculated that OsNRAMP2 could be a novel functional gene associated with Cd accumulation in rice.

     

Divergent evolution of rice blast resistance <Emphasis Type="Italic">Pi54</Emphasis> locus in the genus <Emphasis Type="Italic">Oryza</Emphasis>

Background

The rice blast resistance gene Pi54 was cloned from Oryza sativa ssp. indica cv. Tetep, which conferred broad-spectrum resistance against Magnaporthe oryzae. Pi54 allelic variants have been identified in not only domesticates but also wild rice species, but the majority of japonica and some indica cultivars lost the function.

Results

We here found that Pi54 (Os11g0639100) and its homolog Os11g0640600 (named as #11) were closely located on a 25 kbp region in japonica cv. Sasanishiki compared to a 99 kbp region in japonica cv. Nipponbare. Sasanishiki lost at least six genes containing one other R-gene cluster (Os11g0639600, Os11g0640000, and Os11g0640300). Eight AA-genome species including five wild rice species were classified into either Nipponbare or Sasanishiki type. The BB-genome wild rice species O. punctata was Sasanishiki type. The FF-genome wild rice species O. brachyantha (the basal lineage of Oryza) was neither, because Pi54 was absent and the orientation of the R-gene cluster was reversed in comparison with Nipponbare-type species. The phylogenetic analysis showed that #11gene of O. brachyantha was on the root of both Pi54 and #11 alleles. All Nipponbare-type Pi54 alleles were specifically disrupted by 143 and 37/44?bp insertions compared to Tetep and Sasanishiki type. In addition, Pi54 of japonica cv. Sasanishiki lost nucleotide-binding site and leucine-rich repeat (NBS–LRR) domains owing to additional mutations.

Conclusions

These results suggest that Pi54 might be derived from a tandem duplication of the ancestor #11 gene in progenitor FF-genome species. Two divergent structures of Pi54 locus caused by a mobile unit containing the nearby R-gene cluster could be developed before domestication. This study provides a potential genetic resource of rice breeding for blast resistance in modern cultivars sustainability.

     

The Suppression of Verticillium Wilt of Potato Using Corn as a Green Manure Crop

Field studies involving the effects of growing sweet corn (Zea mays var. Jubilee sweet corn and var. Jubilee super-sweet corn) as a green manure for 2 or 3 seasons demonstrated both suppression of verticillium wilt by 60–70% (Verticillium dahliae Kleb.) and increased potato yields. Although these treatments showed no direct effect on V. dahliae soil populations, the colonization of V. dahliae on potato feeder-roots and in potato tissue of stem apices were reduced. Feeder-root colonization by V. dahliae was positively correlated with verticillium wilt incidence (P?≤?0.05 to P?≤?0.01) and negatively correlated with yield (P?≤?0.05). Corn green manures additionally increased populations of several soilborne fungi which included Ulocladium, and Fusarium equiseti. Specific nutritional and microbial effects were secondary to the effects of cropping practices. When compared with the fallow treatments for 1994, 1995, and 1997, the percentage yield increases for 1994 were: +34% for total yield, +57% for U.S. #1’s, and +127% for tubers >280 g; for 1995 (a year of reduced degree-days and decreased verticillium incidence): +14% for total yields, +15% for U.S. #1 yields, and +21% for tubers >280 g; for 1997: +24% for yield totals, +74% for U.S. #1’s and +179% for tubers >280 g. For establishing these yield benefits, stalks with and without ears of corn were used as green manures. Corn varieties differed for effectiveness as a green manure, which could be accounted for by differences of biomass. When compared with the super-sweet corn, the sweet corn produced an increase (>2-fold) of biomass with less than half of the resulting wilt incidence. When potato was grown consecutively for 2 years, the benefits from green manures became mostly eliminated. However, following 2 consecutive years of potato, a single green manure of sweet corn was sufficient to return the potato crop to the original benefits of verticillium suppression and increased yields. This occurred even though soilborne V. dahliae inoculum levels had increased by >4-fold from 45 to 182 cfu g^?1 of soil. Results of this study demonstrate the importance of green manures and soil-ecology to the management of the Russet Burbank potato.     

Validation of dual-crop coefficient method for calculation of rice evapotranspiration under drying–wetting cycle condition

Based on data collected from rice fields under drying–wetting cycle condition, the procedure of dual-crop coefficient (K _cd) approaches was calibrated and validated to reveal its feasibility and improve its performance in rice evapotranspiration (ET _c) estimation. It was found that K _cd based on FAO-recommended basal crop coefficients (K _cb) underestimated dual-crop coefficients in monsoon climate region in East China. The recommended coefficient (K _cp) value of 1.2 was not high enough to reflect the pulse increase of rice ET _c after soil wetting. The K _cb values were calibrated as 1.52 and 0.63 in midseason and late season, and the K _cp value was adjusted as 1.29 after soil wetting in rice field under drying–wetting cycle condition. The dual-crop coefficient curves based on locally calibrated K _cbCal and K _cpCor matched well with the measured crop coefficients and performed well in calculating rice evapotranspiration from paddy fields under drying–wetting cycle condition. So it can be concluded that the procedure of dual-crop coefficient method is feasible in rice ET _c estimation, and locally calibrated K _cb and K _cp can improve its performance remarkably.     

Identification of resistant germplasm containing novel resistance genes at or tightly linked to the <Emphasis Type="Italic">Pi2/9</Emphasis> locus conferring broad-spectrum resistance against rice blast

Background

The rice Pi2/9 locus harbors multiple resistance (R) genes each controlling broad-spectrum resistance against diverse isolates of Magnaporthe oryzae, a fungal pathogen causing devastating blast disease to rice. Identification of more resistance germplasm containing novel R genes at or tightly linked to the Pi2/9 locus would promote breeding of resistance rice cultivars.

Results

In this study, we aim to identify resistant germplasm containing novel R genes at or tightly linked to the Pi2/9 locus using a molecular marker, designated as Pi2/9-RH (Pi2/9 resistant haplotype), developed from the 5′ portion of the Pi2 sequence which was conserved only in the rice lines containing functional Pi2/9 alleles. DNA analysis using Pi2/9-RH identified 24 positive lines in 55 shortlisted landraces which showed resistance to 4 rice blast isolates. Analysis of partial sequences of the full-length cDNAs of Pi2/9 homologues resulted in the clustering of these 24 lines into 5 haplotypes each containing different Pi2/9 homologues which were designated as Pi2/9-A5, ?A15, ?A42, ?A53, and -A54. Interestingly, Pi2/9-A5 and Pi2/9-A54 are identical to Piz-t and Pi2, respectively. To validate the association of other three novel Pi2/9 homologues with the blast resistance, monogenic lines at BC₃F₃ generation were generated by marker assisted backcrossing (MABC). Resistance assessment of the derived monogenic lines in both the greenhouse and the field hotspot indicated that they all controlled broad-spectrum resistance against rice blast. Moreover, genetic analysis revealed that the blast resistance of these three monogenic lines was co-segregated with Pi2/9-RH, suggesting that the Pi2/9 locus or tightly linked loci could be responsible for the resistance.

Conclusion

The newly developed marker Pi2/9-RH could be used as a potentially diagnostic marker for the quick identification of resistant donors containing functional Pi2/9 alleles or unknown linked R genes. The three new monogenic lines containing the Pi2/9 introgression segment could be used as valuable materials for disease assessment and resistance donors in breeding program.

     

Effective Management of the <Emphasis Type="Italic">Phthorimaea operculella</Emphasis> (Zeller) Using PVA Nanofibers Loaded with <Emphasis Type="Italic">Cinnamomum zeylanicum</Emphasis> Essential Oil

Phthorimaea operculella (Zeller) is one of the most common insect pests of cultivated potato in tropical and subtropical regions. In this research, a potential strategy to improve the insecticidal activity of plant essential oils for the effective management of P. operculella was studied. The insecticidal and residual effects of nanofiber oil (NFO) and pure essential oil (PEO) of Cinnamomum zeylanicum were assessed on PTM under laboratory conditions. The nanofibers were made by the electrospinning method using polyvinyl alcohol (PVA) polymer. The morphological characteristics of the nanofibers were evaluated by scanning electron microscopy and Fourier transform infrared spectroscopy. The chemical constituents of cinnamon essential oil (EO) were detected by GC/MS. Fumigant toxicity of NFO and PEO were evaluated on different growth stages (egg, male and female adults) of P. operculella. SEM and FTIR analyses confirmed the presence of EO on the nanofiber structure. The yield of the EO from C. zelanicum on the nanofibers was 1.86%. GC/MS analysis showed that cinnamaldehyde was the primary constituent (69.88%) of cinnamon EO. LC₅₀ values of C. zelanicum EO and NFO were 4.92 and 1.76 μl/l air for eggs, 0.444 and 0.212 μl/l air for female adults, and 0.424 and 0.192 μl/l air for male adults, respectively. Fumigant bioassays revealed that NFO was more toxic than C. zeylanicum oil against at all stages of P. operculella. The residual effect of PEO and NFO was evaluated against the egg stage of the P. operculella. NFO lost insecticidal effectiveness 47 days after application, while the efficacy of PEO decreased 15 days after application. Our results suggest that NFO of C. zeylanicum can be used as an effective new tool for the management of P. operculella.     

Effects of Different Soil Treatments on the Development of <Emphasis Type="Italic">Spongospora subterranea</Emphasis> f. sp. <Emphasis Type="Italic">subterranea</Emphasis> in Potato Roots and Tubers in the Greenhouse

Powdery scab caused by Spongospora subterranea f. sp. subterranea (Sss) causes extensive losses in potato production systems globally. Two pot experiments were established in the greenhouse in summer 2013 and winter 2014 to evaluate the effectiveness of different soil chemicals, fumigant, amendments and biological control agents (BCAs) against Sss in the rhizospheric soil, potato roots and tubers. The study used visual assessment methods to assess the effect of treatments on root galling and zoosporangia production, and qPCR to measure Sss concentration in the soil and in the potato roots and tubers. All six soil treatments, namely metam sodium, fluazinam, ZincMax, calcium cyanamide, Biocult and a combination of Bacillus subtilis and Trichoderma asperellum recorded significantly (P < 0.05) lower numbers of zoosporangia in the roots compared to the untreated control. The same effect was observed on the concentration of Sss DNA in the roots at tuber initiation. A more diverse picture was obtained when root gall scores at tuber initiation and Sss DNA in the rhizospheric soil at tuber initiation and harvesting were compared. Significant differences (P < 0.05) were also noted in disease severity, disease incidence, and tuber yield between metam sodium, fluazinam, ZincMax, calcium cyanamide and the untreated control. Calcium cyanamide gave the highest tuber yield. The study demonstrated the potential of soil treatments such as metam sodium, fluazinam, ZincMax and calcium cyanamide in managing Sss in potatoes by reducing the pathogen both in the rhizospheric soil and the roots of the potato plant.     

Amplification of the <Emphasis Type="Italic">Phytophthora infestans</Emphasis> RG57 loci Facilitates <Emphasis Type="Italic">in planta</Emphasis> T-RFLP Identification of Late Blight Genotypes

Late blight, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is a devastating disease in potato and tomato and causes yield and quality losses worldwide. The disease first emerged in central America and has since spread in North America including the United States and Canada. Several new genotypes of P. infestans have recently emerged, including US-22, US-23 and US-24. Due to significant economic and environmental impacts, there has been an increasing interest in the rapid identification of P. infestans genotypes. In addition to providing details regarding the various phenotypic characteristics such as fungicide resistance, host preference, and pathogenicity associated with various P. infestans genotypes, information related to pathogen movement and potential recombination may also be determined from the genetic analyses. Restriction fragment length polymorphism (RFLP) analysis with the RG57 loci is one of the most reliable procedures used to genotype P. infestans. However, the RFLP procedure requires propagation and isolation of the pathogen and relatively large amounts of DNA. Isolation of the late blight pathogen is sometimes impossible due to the poor condition of the infected tissues or the presence of fungicide residues. In this study, we describe a procedure to identify P. infestans at the molecular level in planta using terminal restriction fragment length polymorphism (T-RFLP) of the RG57 loci. This T-RFLP assay is sufficiently sensitive to detect and differentiate P. infestans genotypes directly in planta without propagation and isolation of the pathogen, to facilitate the timely implementation of best management practices.