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1.
Raspberry cane disease, caused by a complex of fungi, is amongst the most devastating problems for raspberry production. Using resistant or highly tolerant genotypes is a promising strategy. However, until now, cultivars with good field resistance for German fruit growers as well as the primary causal pathogens of cane disease present in the eastern part of Germany, Saxony state are still unknown. The primary objective of this study was to identify the primary disease causing organisms and resistant Rubus germplasm for future breeding efforts. From 2012 to 2014, we evaluated the degree of susceptibility to cane diseases on 213 raspberry cultivars at two different locations using a rating scale. We also identified the main fungi growing on infected canes. Our results suggest that Fusarium avenaceum is the main fungus causing cane disease in Saxony. The Rubus hybrid cultivars ‘Tayberry’ (2n?=?6x?=?42), ‘Buckingham Tayberry’ (2n?=?6x?=?42) and ‘Dorman Red’ (2n?=?2x?=?14) displayed the highest levels of field resistance at both locations. Moreover, we established an in vitro assay for resistance evaluation which correlates significantly with field data and is particularly suitable for quick assessment of resistance against F. avenaceum in breeding material. Future breeding programs, aiming at cultivars with enhanced resistance towards cane diseases can use the presented data for parental selection and may employ our in vitro F. avenaceum inoculation method for parental testing and progeny selection.  相似文献   

2.
The present study investigated genetic diversity, structure and hybridization in a collection of the endangered wild pear species Pyrus pyraster (L.) Burgd. A total of 278 putative ‘true type’ P. pyraster trees originating from seven populations in the federal state of Saxony in Germany were analyzed along with 35 pear cultivars commonly cultivated in Saxony. The genetic analysis was performed using nine nuclear microsatellite markers (ncSSR) and two paternally inherited chloroplast marker (cpDNA) amplifying in the intergenic spacer region trnQrps16 and the intron region rps16. On basis of the ncSSR dataset after STRUCTURE analysis 80 % of the wild pear individuals were assigned as ‘true type’ P. pyraster genotypes. The cpDNA analysis showed shared haplotypes in P. pyraster and P. communis but with an unequal frequency in both species. The analysis of molecular variance resulted in a moderate (ncSSR) and great (cpDNA) variation among ‘true type’ P. pyraster and the pear cultivars. The genetic diversity in the ‘true type’ P. pyraster populations was still high and the genetic structure between the populations low (ncSSR and cpDNA) indicating a genetic exchange between the populations by pollen and seeds. The clear discrimination between the P. pyraster and P. communis confirms our expectation of the existence of ‘true type’ P. pyraster individuals in the study area. The existing genetic integrity and the high genetic diversity argue for the implementation of preservation measures in P. pyraster.  相似文献   

3.
According to Pliny the Elder and other Greco-Roman geoponics, Raetica was a famous white grape as well as a white wine produced in Raetia, a Province of the Roman Empire. Does Raetica grape have modern descendants? Etymologically and geographically, the white ‘Rèze’ from Valais (Switzerland) would be the best candidate. Using available microsatellite data, we searched for relatives of ‘Rèze’ in our database containing over 1,700 genotypes of grape cultivars from all over the world. Twelve cultivars showing putative first-degree (parent–offspring or full-siblings) or second-degree (grandparent–grandoffspring, uncle–nephew or half-siblings) relationships with ‘Rèze’ were then analysed at 60 microsatellite markers. Calculation of allele sharing and likelihood ratios between competing relationship categories revealed that four cultivars had parent–offspring relationship with ‘Rèze’: ‘Cascarolo Bianco’ (Piedmont, Italy), ‘Arvine Grande’ (Valais, Switzerland), ‘Groppello di Revò’ and ‘Nosiola’ (Trentino, Italy). Given that some of these are also said to be Raetica descendants, we may well be on the tracks of Pliny the Elder’s Raetica grape. However, there is no evidence about the identity of Raetica. Analysis of ancient DNA of grape pips excavated from archaeological sites of the Roman times might provide key information. Our first attempts were unsuccessful, but analysis of additional samples and optimisation of the method could provide groundbreaking results about the identity of the grapes cultivated in classical antiquity.  相似文献   

4.
Analysis of the genetic structure of Indonesian Oryza sativa and O. rufipogon using neighbour-joining trees based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers revealed that O. sativa in Indonesia is separated from O. rufipogon. Accessions of O. sativa in this study were differentiated into two major groups, indica and tropical japonica, excluding some varieties. SSR and SNP markers revealed the high value of differentiation (F ST) and genetic distance (D) between indica and tropical japonica and we discovered four loci by SNP markers and one locus by SSR markers that play a role in differentiation between indica and tropical japonica. Interestingly, genetic diversity (H) in O. rufipogon was lower than that in O. sativa, however H in O. rufipogon was the highest and H in tropical japonica was the lowest when O. sativa was divided into two groups. Inbreeding coefficient (Fst) showed evidences that gene flow (Nm) between species and within species might be one of the mechanisms related to the diversification and differentiation of Indonesian rice germplasm by asymmetric pattern between species and within O. sativa as revealed by SSR and SNP markers. In addition, we found evidences on stabilizing selection in Indonesian rice germplasm and they might be the reasons why Indonesian rice germplasm did not differentiate due to source location of landrace. However, we found a weak relation between SSR and SNP markers probably due to highly polymorphic in SSR and the different properties of both markers.  相似文献   

5.
Yam bean [Pachyrhizus DC.] is a legume genus of the subtribe Glycininae with three root crop species [P. erosus (L.) Urban, P. tuberosus (Lam.) Spreng., and P. ahipa (Wedd.) Parodi]. Two of the four cultivar groups found in P. tuberosus were studied: the roots of ‘Ashipa’ cultivars with low root dry matter (DM) content similar to P. erosus and P. ahipa are traditionally consumed raw as fruits, whereas ‘Chuin’ cultivars with high root DM content are cooked and consumed like manioc roots. Interspecific hybrids between yam bean species are generally completely fertile. This study examines the genetic diversity of the three crop species, their potentials for breeding and the identification of useful traits to differentiate among yam bean genotypes and accessions. In total, 34 entries (genotypes and accessions) were grown during 2000?2001 at two locations in Benin, West Africa, and 75 morphological and agronomical traits, encompassing 50 quantitative and 25 qualitative characters were measured. Diversity between entries was analyzed using principal component analysis, cluster analysis, multivariate analysis of variance and discriminant function analysis. Furthermore, phenotypic variation within and among species was investigated. Intra- and interspecific phenotypic diversity was quantified using the Shannon–Weaver diversity index. A character discard was tested by variance component estimations and multiple regression analysis. Quantitative trait variation ranged from 0.81 (for total harvest index) to 49.35% (for no. of storage roots per plant). Interspecific phenotypic variation was higher than intraspecific for quantitative traits in contrast to qualitative characters. Phenotypic variation was higher in overall for quantitative than qualitative traits. In general, intraspecific phenotypic variation ranged from 0.00 to 82.61%, and from 0.00 to 80.03% for quantitative and qualitative traits, respectively. Interspecific phenotypic variation ranged from 0.00 to 95.02%, and 0.00?81.58% for the two trait types, respectively. The Shannon–Weaver diversity index (H′) was in general high and over 0.80 for most of the trait. Diversity within P. tuberosus was higher than within P. erosus and P. ahipa. Across the 50 quantitative and 25 qualitative traits, the Shannon–Weaver diversity index of intra- and interspecific variation was around 0.83 and 0.51, respectively and was lower for qualitative than for quantitative traits. Monomorphism was observed in eight qualitative traits and one quantitative character. The first, second and third principal components explained, respectively, 39.1, 21.3 and 8.3% of the total variation in all traits. Pachyrhizus erosus, P. ahipa, and P. tuberosus (‘Chuin’ and ‘Ashipa’) were clearly separated from each other by these analyses. Multivariate analysis of variance indicates significant differences between Pachyrhizus species for all individual or grouped traits. Discriminant function analysis revealed that the first two discriminant functions were almost significant. Biases due to unbalanced sample size used per species were small. Within each species a similar amount of diversity was observed and was determinable to 70% by only ten traits. We conclude that the cultivated yam bean species represent distinct genepools and each exhibits similarly large amounts of genetic diversity.  相似文献   

6.
Some more than a century old Chinese fir genotypes with high biomass that are now rare in China were historically called “the king of Chinese fir”. The genetic diversity and relationships of those ancient Chinese fir genotypes were investigated using morphological analysis and sequence-related amplified polymorphism markers. The morphological analysis indicated that the tree volume parameter had the maximum variable coefficient value (70.2 %). In total, 18 selected primers generated 154 bands, and 150 bands were polymorphic (97.4 %). Higher values of genetic diversity parameters (h = 0.3593, I = 0.5283) were maintained at the species level, indicating that the ancient Chinese fir in China has retained a relatively high level of genetic diversity. AMOVA indicated that 73.16 % of the variation resided within provenances. The UPGMA dendrogram and genetic structure analysis identified 3 major clusters and grouped the genotypes in agreement with their geographic origins. A Mantel test revealed a significant correlation between genetic distances and geographic distances among the genotypes (r = 0.4275, p < 0.01). Overall, we recommend a combination of conservation measures including a germplasm repository and the implementation of in situ conservation.  相似文献   

7.
We investigated the genetic variation and relationships among 35 melon landraces collected from the Xinjiang Uygur Autonomous Region in northwestern China by using 19 polymorphic simple sequence repeat markers (SSRs). A total of 55 polymorphic alleles were amplified. The number of alleles per SSR locus ranged from 2 to 5 with an average of 2.89 alleles per locus. The average gene diversity (GD) was 0.42 with a range of 0.06–0.71, and the average observed heterozygosity was 0.22 with a range of 0.06–0.97, indicating that the genetic diversity among the Xinjiang melon landraces was abundant. Genetic variation was also detected between the landrace populations in different regions in Xinjiang. The most abundant genetic diversity was observed among the landraces in Eastern Xinjiang, with the highest GD of 0.45 and PIC of 0.39. Eleven alleles (20 %) were found exclusively in the landraces from Eastern Xinjiang, and two alleles (3.6 %) were unique to the landraces from Southern and Northern Xinjiang. The genetic similarity matrix was defined on the basis of Jaccard coefficient to determine the genetic relationships among Xinjiang landraces. Cluster analysis was performed using the unweighted pair group method with arithmetic means, showing that the ‘wild Hami’ (XJ-34) landrace was distinct from the 34 other landraces that were divided into three clusters. Therefore, the genetic background of XJ-34 differed from that of the other landraces. The landraces were not precisely separated on the basis of their geographic origins, although most of these landraces were likely grouped near one another, as visualized through principal coordinate analysis. Thus, western China is one of the primary or secondary centers of melon diversity because of the relatively higher genetic variation detected among Xinjiang landraces. Except the ‘wild Hami’ landrace, Xinjiang melon landraces could be classified into two botanical varieties, namely, var. inodorus and var. cantalupensis. However, the distinction between these two genotypes was not significantly different.  相似文献   

8.
We report here that small but numerous foliar glands are present near the base of the abaxial leaf blades of hop (Humulus lupulus L.) at about 2.6 times the density found in the more distal regions, and this concentration of glands appear to protect the basal region from damage by insects. Distinctive wild taxa of H. lupulus are native to distinctive regions of the north temperate world. The much higher presence of protective glands in the three native North American Humulus taxonomic varieties in comparison with the varieties native to Europe and Asia suggests specialized leaf feeders in North America have been responsible for the evolution of greater gland density. Commercial domesticated hop cultivars trace most of their germplasm to the European hop (H. lupulus var. lupulus), which possesses a much lower concentration of leaf glands than the native hops of all other regions of the world, and therefore is likely relatively susceptible to damage. We suggest that breeding for increased density of protective foliage glands may be a non-chemical way of alleviating foliar feeding damage in cultivated hops.  相似文献   

9.
Developing a molecular tool kit for hybrid breeding of Osmanthus species and related genera is an important step in creating a systematic breeding program for this species. To date, molecular resources have been aimed solely at Osmanthus fragrans with little work to develop markers for other species and cultivars. The objectives of this study were to (1) determine cross-transferability of O. fragrans and Chionanthus retusus derived SSRs in diverse Osmanthus taxa, (2) quantify the influence of locus-specific factors on cross-transferability, and (3) determine the genetic relationships between accessions. We tested 70 SSR markers derived from O. fragrans and C. retusus in 24 accessions of Osmanthus. Sixty-seven markers showed transfer to at least one other Osmanthus species with an overall transfer rate of 84% of loci across taxa. Genotyping with 42 microsatellite markers yielded a total of 367 loci. Number of alleles per locus ranged from 2 to 17 with a mean of 8.7 ± 4.8. Mean observed and expected heterozygosities were 0.560 ± 0.225 and 0.688 ± 0.230, respectively. Percent of polymorphic loci ranged from 40% in Osmanthus delavayi to 100% in O. fragrans. Osmanthus fragrans had the highest mean number of alleles per locus (4.2) while O. delavayi had the lowest (1.1). A reduced suite of eight-markers can distinguish between accessions with non-exclusion probabilities of identity from 3.91E?04 to 2.90E?07. The SSR markers described herein will be immediately useful to characterize germplasm, identify hybrids, and aid in understanding the level of genetic diversity and relationships within the cultivated germplasm.  相似文献   

10.
Momordica cochinchinensis (Cucurbitaceae) is native to South East Asia and is important for its nutritional and medicinal properties. There is little information available on morphological and genetic diversity, crop selection and strategic germplasm collection for M. cochinchinensis. In this study, 42 accessions of M. cochinchinensis collected from Vietnam, Thailand and Australia were analysed for diversity study both at a morphological and molecular level. Both morphological and molecular analyses were in agreement and clustered the accessions based on the country of origin. The markers investigated in this study could be used for future applications with samples from unknown origins or for gene tracking for important cultivars. The Momordica accessions were highly polymorphic based on the markers suggesting high genetic diversity. Therefore the results from this study will be important in future strategic germplasm collection, conservation, horticultural development and commercial propagation of M. cochinchinensis.  相似文献   

11.
Nine genic SSR loci were used to evaluate the genetic diversity and identify accessions in wild Italian Humulus lupulus L., in comparison with widely cultivated European and U.S. commercial cultivars. A collection of 80 wild hop samples from Italy and 43 hop cultivars from Europe and U.S., were characterized. Allelic frequency analysis revealed 65 distinct Italian genotypes and differentiated all the commercial cultivars; moreover, specific alleles were observed for wild and cultivated hops. The number of alleles identified in the wild population were 104 and 123 within all the accessions. The maximum polymorphic information content was evidenced for locus HlGA23 in the Italian wild population and in the whole set of accessions (0.905 and 0.902 respectively). The dendrogram constructed from Euclidean distance with the UPGMA method showed two main clusters, one including commercial American and European accessions and one mostly composed by wild Italian accessions. Model-based clustering (Bayesian method) placed the accessions into five germplasm groups, one of which was characterized by Italian genotypes only. The study showed for the first time the great biodiversity present in Italy, and the remarkable differences with European and American hops. It was also found that within the population of north-central Italy a large genetic variability is present, suited to be studied and exploited; this genetic wealth could be used in future breeding programs in order to develop new hop varieties carrying characteristics useful for brewers.  相似文献   

12.
The present study used 15 simple sequence repeat loci to characterize the genetic diversity of the germplasm that originated the current industrial chicory and to establish the relationships between and inside Cichoriumintybus L. and Cichorium endivia L. Initially we analyzed 19 cultivated C. endivia accessions, 27 wild and 155 cultivated C. intybus accessions distributed among three groups: 83 root chicories, 42 Witloof and 30 leaf chicories. The leaf chicories comprised cultivars corresponding to the Radicchio, Sugarloaf and Catalogne subgroups. The latter has not been previously included in any genetic diversity study. Subsequently, 1297 individuals from the 15 modern root chicory cultivars at the origin of the breeding of the current industrial root chicory cultivars were analyzed. Although the accessions of C. endivia and C. intybus were clearly separated from each other, seven wild C. intybus individuals were genetically closer to C. endivia than to C. intybus, revealing complex genetic interrelationships between these species. The differentiation of C. intybus into three cultivar groups (Witloof, root chicory and leaf chicory) was confirmed. The leaf chicory individuals were divided into three genetic subgroups, corresponding to the Radicchio, Sugarloaf and Catalogne cultivars, thus attesting to the validity of the classification based on morphological factors. Clear differentiation was observed among the Belgian, Polish and Austrian modern industrial root cultivars, but not among the French industrial modern root cultivars. The high phenotypic and genetic variability of the modern industrial root cultivars indicates that this germplasm constitutes a useful gene pool for cultivar improvement and selection.  相似文献   

13.
14.
Forty-four Asparagus officinalis cultivars, gene bank accessions and breeding lines as well as thirty-four accessions of wild relatives of Asparagus were evaluated for resistance to Asparagus virus 1. Three different test strategies were developed for the assessment of individual plants: (1) natural infection under field conditions, or two vector-mediated infection assays using the green peach aphid Myzus persicae (2) in an insect-proof gauze cage or (3) in a climate chamber. The AV-1 infections were verified by DAS-ELISA and RT-PCR approaches. All tested 660 individual plants of A. officinalis germplasm were susceptible to AV-1 infection. In contrast, in 276 plants of 29 Asparagus wild accessions no virus infection could be detected. These resistant accessions comprised of nineteen diploid, tetraploid and hexaploid species of both the Eurasian clade and the African clade of the asparagus germplasm. Data of the AV-1 resistance evaluation are discussed in relation to the genetic distance of the resistance carrier and potential application in breeding.  相似文献   

15.
Genetic variations and relationships among cultivated and wild genotypes of five taxa of Indian Luffa were examined using inter simple sequence repeats (ISSR), directed amplification of minisatellite DNA (DAMD) markers and morphological characterization. Morphometric evaluation of 21 discrete characters in 51 representative accessions segregated the five taxa of Luffa in three main clusters: the two wild species (L. echinata, L. graveolens) in the first, and the cultivated L. aegyptiaca (smooth gourd) and L. acutangula (ridged gourd)/L. hermaphrodita (Hermaphrodite luffa) in the second and third clusters, respectively. Cumulative data analysis of 15 ISSR and seven DAMD markers revealed high percentage polymorphism (97.67 %), moderate genetic distance (0.06–0.72, avg. 0.51), and low heterozygosity and Shannon index values (H = 0.15; I = 0.22) across all the 76 genotypes assayed. A UPGMA dendrogram, based on the combined marker data, resolved the five taxa in two main clusters with high bootstrap support. The morphological and molecular trees showed incongruence in the number of main clusters resolved and in the disposition of the wild and cultivated taxa in different sub-clusters. The cluster analyses and PCoA plots revealed a nested grouping of the hermaphrodite luffa within the ridge gourd group. The Bayesian STRUCTURE analysis identified three genetic clusters for the five assumed taxa. Outcrossing test revealed a mixed mating system in Indian Luffa. This is the first ever report on the mating system in Luffa using molecular markers. The study also demonstrates the utility of using more than one DNA marker in the assessment of molecular diversity in a widely cultivated crop genus like Luffa with a narrow genetic base.  相似文献   

16.
Cañahua (Chenopodium pallidicaule Aellen) is a poorly studied, annual subsistence crop of the high Andes of South America. Its nutritional value (high in protein and mineral content) and ability to thrive in harsh climates make it an important regional food crop throughout the Andean region. The objectives of this study were to develop genetic markers and to quantify genetic diversity within cañahua. A set of 43 wild and cultivated cañahua genotypes and two related species (Chenopodium quinoa Willd. and Chenopodium petiolare Kunth) were evaluated for polymorphism using 192 microsatellite markers derived from random genomic cañahua sequences produced by 454 pyrosequencing of cañahua genomic DNA. Another 424 microsatellite markers from C. quinoa were also evaluated for cross-species amplification and polymorphism in cañahua. A total of 34 polymorphic microsatellite marker loci were identified which detected a total of 154 alleles with an average of 4.5 alleles per marker locus and an average heterozygosity value of 0.49. A cluster analysis, based on Nei genetic distance, clearly separated from wild cañahua genotypes from the cultivated genotypes. Within the cultivated genotypes, subclades were partitioned by AMOVA analysis into six model-based clusters, including a subclade consisting sole of erect morphotypes. The isolation by distance test displayed no significant correlation between geographic collection origin and genotypic data, suggesting that cañahua populations have moved extensively, presumably via ancient food exchange strategies among native peoples of the Andean region. The molecular markers reported here are a significant resource for ongoing efforts to characterize the extensive Bolivian and Peruvian cañahua germplasm banks, including the development of core germplasm collections needed to support emerging breeding programs.  相似文献   

17.
A barley core collection can be studied extensively and the derived information can be used to identify loci/genes for the genetic improvement of quantitative and qualitative traits. To assess genetic diversity, allelic variation and population structure of Egyptian barley, 134 barley genotypes collected from a different region along with 19 cultivated genotypes obtained from of the Egyptian Agricultural Research Center. All genotypes were analyzed with 261 polymorphic SSR and SNP alleles. The mean number of alleles per locus was 4 and PIC was 0.49, while the level of genetic diversity was 0.55 ranging from 0.03 to 0.82. The genotypes were assigned to three subpopulations that were consistent with their origins. The genetic variation within population was higher (51%) than among population at the molecular levels (FST =?0.491 when P?<?0.10). The level of polymorphic variation was highest in subpopulations-II, due to collected from different regions with different ear-types thus, expected to contain more diversity than local genotypes in subpopulations-I and subpopulations-III. The structured study found that the 153 barley genotypes are in harmony with clustering approaches using the SSR and SNP genotypic data in a neighbor-joining tree and principal components analysis, which identified three subpopulations. These results demonstrated genetic diversity among the Egyptian barley genotypes can be applied to suggest approaches, such as association analysis, classical mapping population development, and parental line selection in breeding programs. Therefore, it is necessary to use the exotic genotypes as the genetic resources for developing new barley cultivars in Egypt.  相似文献   

18.
Phenolic acid intake through the consumption of whole-wheat foods provides important health benefits associated with reduced risks of cardiovascular diseases and colon cancer. The genetic variation for phenolic acids was extensively studied in common wheat, but a comprehensive survey in tetraploid wheat is lacking. In this study we evaluated the genetic variability for individual and total phenolic acids concentration existing in a large collection of tetraploid wheat (Triticum turgidum L.). A 2-year evaluation was undertaken on the whole-meal flour of 111 genotypes belonging to seven T. turgidum subspecies including cultivars, landraces and wild accessions. Durum cultivars [T. turgidum subsp. durum (Desf.) MacKey], had the highest average concentration of total phenolic acids (828.7 μg g?1 dm in 2012; 834.5 μg g?1 dm in 2013) with amounts varying from 550.9 μg g?1 dm to 1701.2 μg g?1 dm, indicating a variation of greater than threefold fold. The lowest concentration of phenolic acids was found in T. turgidum subsp. dicoccum (Schrank ex Schübler) Thell. Rivet wheat (T. turgidum L. subsp. turgidum) had phenolic acid concentrations similar to those in durum, but less variation was noted among the accessions. On the other hand, the accessions of the four remaining subspecies showed lower phenolic acid concentrations and variation among the accessions as compared to durum. A total of six phenolic acids were identified across the wheat genotypes. The effects of genotype, year and year × genotype were estimated by ANOVA and resulted significant for all phenolic acids. The ratio of genotypic variance to total variance suggested the possibility of improving phenolic acid content in elite wheat germplasm through appropriate breeding programs. Moreover, significant correlations between phenolic acids and other quality characteristics of the grain were detected.  相似文献   

19.
Insecticidal crystal (Cry) proteins produced by transgenic Bacillus thuringiensis (Bt) rice that enter the soil via root exudation and plant residues may be harmful to non-target soil organisms. We conducted a 3-year field investigation to determine if soil nematode abundance and diversity were affected by exposure to two transgenic Bt rice cultivars, compared to their non-transgenic near isolines. Near isolines were Kemingdao (KMD-Bt) expressing the single Cry1Ab gene and its non-Bt near isoline Xiushui-11 (XSD), as well as Huahui-1 (HH-Bt) expressing the fused Cry1Ab/Cry1Ac gene and its non-Bt near isoline Minghui-63 (MH). Nematode variables including community composition, abundance, trophic groups, and most of the common genera differed significantly between the rice cultivars. However, these nematode variables were similar under transgenic Bt rice and its non-Bt near isoline, although higher Shannon’s index value and Pielou’s index value were found in soils planted with Bt rice than the non-Bt near isoline. During this 3-year field study, gene modification (single Cry1Ab gene and fused Cry1Ab/Cry1Ac gene) supports a more uniform distribution of nematode species but had no effect on soil nematode abundance and community composition. We conclude that continuous cultivation of KMD-Bt and HH-Bt rice varieties for 3 years is not detrimental to soil nematode communities under field conditions.  相似文献   

20.
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