Identification Of The Important Plant Pests And Study On Their Molecular Systematics

Pest invasion has been becoming a hot research field all over the world, because it has been the second most severe threat to biodiversity. China has grown up to be the fourth largest importer and the fifth largest exporter of agricultural trade, and one of the countries which are affected most by biological invasion. As the further expansion of foreign trade, import and export of plants and their products have become more and more frequent. Then, the controlling of invasive species has been becoming an important and urgent issue which is severe hazards to biological diversity, ecological environment and the national economy. To solve this big issue, the first barrier is to strengthen customs inspection and quarantine to protect the safety of agricultural production. Therefore, new techniques with higher accuracy and efficiency developed for this purpose will be great practical significance to our agricultural domestic trade and export.In recent years, with the need of urban landscape and animal husbandry, a large quantity of Gramineae grass seeds has been imported from abroad, especially bluegrass, ryegrass, brome, red fescue and bentgrass, which are one of the most important hosts of smut fungi. The yield and quality of agriculture products are seriously affected by Tilletia fungi, of which wheat dwarf bunt Tilletia controversa is a quarantine pest prohibited to enter China. T. controversa endangers the wheat and infects many Gramineae grass seeds which may carry Tilletia species without occurring in my homeland. As soil-borne and seed-infected fungi pathogens, Once introduced it will be very difficult to prevent and eradicate them. To strengthen the port inspection and quarantine to smut fungi, we developed a more sensitive and efficient technique—triple PCR—to detect three Tilletia species infecting ryegrass T. controversa,T. lolii and T. vankyi which are very similar in reticulate-spored morphology. This technique can be applied to detect three smut fungi at the same time, which significantly reduces the cost in distinguishing them. Also, the molecular diagnostic methods were developed to identify T. bromi on brome grasses and T. vankyi on red fescue by using the DNA samples from either the mycelia or the teliospores isolated from grasses. Novel techniques of double PCR for hyphal genomic DNA and nested-double PCR for teliospores were developed to identify T. sphaerococca and TCT on bent grasses. Furthermore, the Novel techniques of double PCR and nested-double PCR were applied to successfully distinguish the T. sp. and T. fusca on bluegrasses. Moreover, we did the phylogenetic analysis based on the Tilletia IGS1 gene, which shows that T. sp. has closer genetic distance to T. Bromi, but farther genetic distance to T. fusca relatively. The morphological and physiological characteristics of germination of teliospores of two new Tilletia species T. vankyi and T. puccinelliae were studied and the results were brought into two papers about the two new species. And the phylogenetic analysis based on the Tilletia IGS1 gene were carried out to characteristic the two new species. Our results showed that they are consistent with the conclusion from two papers about the two new species. In conclusion, the identification protocols are successfully developed to identify the smut fungi from these five Gramineae grasses.Soybean (Glycine max L. Merrill) is a very important crop and has been imported into China on a large scale recently. Bean pod mottle virus (BPMV, Genus: Comovirus, Family:Comoviridae) is an important seedborne virus in soybean, causing quality and yield loss seriously due to seed coat mottling and seed weight reduction. Therefore, it is an important quarantine pest for China. BPMV is not original from China, but has been intercepted frequently from imported soybean from USA. For detection of BPMV in imported soybean, the specific primers and Taqman probe were designed based on the coding gene of BPMV coat protein (cp) and one step RT-PCR and one step real-time fluorescent RT-PCR methods were developed. The aplicon of one step RT-PCR was about 500bp, and the detection limit for real-time fluorescent PCR was 20fg/μL of positive cloning plasmid, while that was 100-fold higher than that of RT-PCR, and the detection duration shortened from at least 8h to 4h. The quick, sensitive, accutate and convenient detection method of one step real-time fluorescent RT-PCR for BPMV has been successfully established in this study and it is adaptive greatly to port detection. In addition, the relationship between hilum pigmentation and the virus infection was carried out with above molecular method and DAS-ELISA assay. Our results suggest that the color of hilum is correlated with virus infection, and BPMV was detected from soybean with brown hilum, but not black hilum. Soybean mosaic virus (SMV) was detected from soybean with black, not brown hilum. Also, other eight viruses were not detected from the soybean with brown hilum with DAS-ELISA test. The phylogenetic analysis based on the BPMV cp gene was carried out to study the strain differentiation. The result suggests that the BPMV isolates infected imported soybean from USA may be belong to subgroup II.The fall webworm, Hyphantria cunea Drury (Lepidoptera:Arctiidae), is a severe invasive and quarantine pest all over the world which has a wide range of habitats. It is a polyphagus pest that feeds on about 175 species of broad leaf trees in China, which has caused serious damage to forest trees throughout its range and appears to be continuing to spread. It also damages the roadside and garden trees around urban areas. Mitochondrial genome (mitogenome) is usually used for determining population structure, phylogenetic relationships and general evolutionary events because of its small genome size, stable gene content, uniparental inheritance, and lack of extensive recombination and the accelerated rate of nucleotide substitution. However, limited mitogenomes, representing only a few families in this species-rich insect order, have been sequenced. Therefore, characterization of more lepidopteran mitogenomes is needed to address evolutionary questions and traits, and to probe into the phylogenetic relationships among the lepidopteran superfamilies. In this study, the complete mitogenome of the fall webworm, Hyphantria cunea (Lepidoptera:Arctiidae) was determined. The genome is a circular molecule 15 481bp. It presents a typical gene organization and order for completely sequenced lepidopteran mitogenomes. As found in all sequenced lepidopteran mitogenomes, by the translocation of tRNAMet to a position 5'upstream of tRNAIle, the placement of tRNAMet of H. cunea differs from that of Drosophila yakuba, the hypothesized ancestral gene order of insects. The nucleotide composition of the genome is also highly A+T biased, accounting for 80.38%, with a slightly positive AT skewness (0.010), indicating the occurrence of more As than Ts, as found in the Noctuoidea species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI, which is tentatively designated by the CGA codon as observed in other lepidopterans. Four of 13 PCGs harbor the incomplete termination codon, T or TA. All tRNAs have a typical clover-leaf structure of mitochondrial tRNAs, except for tRNASer (AGN), the DHU arm of which could not form a stable stem-loop structure. The intergenic spacer sequence between tRNASer(AGN) and ND1 also contains the ATACTAA motif, which is conserved across the Lepidoptera order. The H. cunea A+T-rich region of 357bp is comprised of non-repetitive sequences, but harbors several features common to the Lepidoptera insects, including the motif ATAGA followed by an 18bp poly-T stretch, a microsatellite-like (AT)8 element preceded by the ATTTA motif, Finally, an llbp poly-A present immediately upstream tRNAMet. The phylogentic analyses of the available lepidopteran species support view that the H. cunea is closer related to the Lymantria dispar than Ochrogaster lunifer, and support the hypothesis that Noctuoidea (H. cunea, L. dispar, and O. lunifer) and Geometroidea (Phthonandria atrilineata) are monophyletic. However, in the phylogenetic trees based on mitogenome sequences among the lepidopteran superfamilies, Papillonoidea (Artogeia melete, Acraea issoria, and Coreana raphaelis) joined basally within the monophyly of Lepidoptera, which is different to the traditional classification.In the genus Sorghum, there are some important grain crops and economically and ecologically important forage grasses together with three agricultural quarantine weed pests which are S. halepense, S. almum and S. silk. In the present study, Total DNA from the seeds of eight plant materials was extracted, and the Adh1 gene analogues about 2 OOObp long were obtained by PCR strategy using specific primers designed from conserved regions of Adh1 gene reported in the GenBank (AF050456). All PCR products were cloned and sequenced. Based on these sequences and 24 sequences registered in the GenBank, the phylogenetic trees constructed by multiple methods (MP, ML and NJ) supported nearly the same topology. Our molecular results show that:(1) there are obviously three lineages for genus Sorghum. One including two subgenera Chaetosorghum and Heterosorghum, and the second being subgenus Eusorghum, consist of 2n=20 and 2n=40 species with small chromosomes, and the third containing two subgenera Parasorghum and Stiposorghum comprises 2n=10 species and their polyploid relatives with relatively large chromosomes; (2) In subgenus Eusorghum, three quarantine weed pests S. halepense, S. almum and S. silk stay together with morphologically similar species such as S. bicolor and its subspecies S. arundinaceum, S. sudanense and S. propinquum, which is in accordance with the results of morphological study. And S. silk is closer to S. sudanense and S. almum. (3) there lies geographical divergence between the S. almum populations obviously; (4) S. purpureosericeum, S. versicolor, S. nitidum and S. leiocladum belonging to subgenus Parasorghum clust together, but S. matarankense, S. grande and S. timorense belonging to the same subgenus clust with the species of subgenus Stiposorghum and have shown closer relationship to subgenus Stiposorghum; (5) S. macrospermum shows more closely related to S. laxiflorum than the other species of the genus Sorghum. These relationships between Sorghum species provide an important guide for plant breeders to exploit wider Sorghum genepool through crosses between wild and cultivated species in an effort to improve Sorghum production, and are helpful in port detections of the three quarantine weed pests.In this study, molecular detection techniques were developed to detect smut fungi on five kinds of imported Gramineae grasses, and BPMV infecting soybean. These techniques could be applied to port-related species and trains sreening, early diagnosis of diseases of the domestic field and dynamic monitoring of the epidemic in agriculture. And molecular systematic analyses of reticulate-spored Tilletia spp, BPMV, fall webworm and three quarantine weeds in sorghum were carried out. These pests have interceptd regularly in port detection and so have caused a real threat to agriculture. Our results help to clarify the molecular level phylogenetic relationships among species and strain differentiation of virus, not only of great theoretical significance, also of practice significance in plant protection in agriculture. Our techniques and results have important applications in clarifying taxonomic status, supplementing and improving the traditional morphological classification at molecular level. This study will improve our agricultural plants against harmful alien invasion and spread of epidemic and quarantine monitoring capabilities. Also it can improve agricultural inspection and quarantine of plant quarantine pests overall level of agricultural production to ensure safety and promote the smooth export of agricultural products. This has important political significance and economic significance to our agriculture development.