| Citrus Huanglongbing (HLB), also called citrus greening disease is the most destructive and devastating disease of citrus in the world, No effective controls of citrus greening disease.Now. Diaphorina citri is only a insect vector tranmitted HLB. Murraya paniculata is Rutaceae, is made for host.also is host of hlb Murraya paniculata infrequently shown symptom after infected hlb, it was studied as a resistence material to HLB. there is a little paper on the status of gene express infected HLB.In this paper, some field samples were collected with typical symptom of HLB, identification of biology chacter and PCR was carried in the laboratory. Experiments under nature environment were conducted to the host preferences of Diaphorina citri to Carmona microphylla andvarious hosts of Rutaceae. The life cycle on the C. microphylla and Murraya paniculata were recorded and analyzed under the natural conditions. The tolerance to starvation and thirstiness of different aged nymphs and adult D. citri were tested in the laboratory condition with the situation in lack of food and water. Different concent rations of ethanol extract of Alocasia macrorrhizos leaves.were used to deal with larvae and adults of Diaphorina citr..i. Folders micro-poisonous leaves method were measured.Different transmission methods that Candidatus Liberibacter asiaticus infected Murraya paniculata, were compared and Nested-PCR was used to confirm the presence of Ca. Liberibacter asiaticus two months after inoculation. Complex cDNA library of Murraya paniculata with infected Citrus HuangLongBing at various times was constructed using suppression subtractive hybridization (SSH). according to EST from SSH library On Murraya paniculats pathogenesis-related protein STH-21 gene (accession numberU21849), using hiTAIL-PCR technology, a full-length cDNA of pathogenesis-related protein named sth- 21 was obtained from Murraya paniculats. A semi RT-PCR system was established to detect the gene expression of miraculin-like protein, sthp2-21, ubiquitin-conjugating enzyme 8, chlorophyll A/B binding protein in Murraya paniculata with HLB in the earlier stages.1 The results shows Diaphorina citri can survive on Carmona microphylla, With development duration of one generation Diaphorina citri fed on Carmona microphylla hosts varied significantly, with the long nymphaes ages(15-20d) and the adult nymphaes ages(17-19d).as contrast host., Murraya paniculata,15.0～17.0d and 20.0～23.0d respectively., and the life cycle in Carmona microphylla was close to Murraya paniculata, while the surviving rate is lower.about 16.9%. Carmona microphylla is an actetable host, rather than an optimum host. The results showed that the tendency fed on different hosts varied significantly, Murraya paniculata and clausena were favorite host for Diaphorina citri, Citrus. limon is inferior to the former, There were no significant differences between Citrus sinensis Osbeck and C. grandis (L) Osbeck and Citrus reticulata Blanco cv. Ponkan. Diaphorina citri seldom feed on Poncirus trifoliata (L) Raf.. The starvation and thirstiness tolerance of adults was described as the mean survival time. Thev results showed that in the three treatments as mentioned, the mean survival time of young and old nymphae age and adults were7.7～8.2h,15.0h～18.0h,36.0h～38.0h respectively when no food and water..10.0h～12.0h and35.0h～37.0h and 48.0h～50.0h respectively when providing food without water.11.0h～13.0h and 39.0h～40.0h and 69.0h～75h.0h when providing water without food. Water would prolong the mean survival time of adults significantly, starvation tolerance and thirstiness tolerance of adults adults were stronger than nymphae ages. Results indicated that all levels of the concent rations of ethanol extract have different levels of killing effect. The corrected mortality were 45.6%,98.4%,23.5%,71.0%,3.89%,8.5% and 11.5% by the 3 concentrations of Alocasia macrorrhizos treated with 10g/100mL,1g/100mL,0.1 g/100mL extract of ethanol, Volatile ingredients of Alocasia macrorrhizo has strongly killing effect at 0.5h。2 The results indicated that mechanical inoculation, injection and squeezing methods failed to transmit Ca. Liberibacter asiaticus to Murraya paniculata, whereas grafting from infected Hongjiang Sweet Orange (Citrus sinensis Osbeck) was successful in spite of no scions elongation, and both dodder (Cuscuta campestris) and psyllid (Diaphorina citri) were able to successfully transmit HLB bacterium from citrus to Murraya paniculata. One psyllid was able to transmit it successfully with the shortest time of psyllid feeding on Hongjiang Sweet Orange,1.5h to 2h, and 12h for Diaphorina citri to transmit HLB bacterium to Murraya paniculata, respectively. Efficient temperatures for HLB transmission by psyllid were between 15℃and 35℃, and there were no transmission of HLB bacterium below 0℃and over 40℃. The study laid a foundation for the large scale screening of resistant-related gene to Ca. Liberibacter asiaticus from Murraya paniculata.3 Two differentially expressed genes libraries-a library of up-regulated genes and a library of down-regulated genes in HLB-infected Mexican lime (C. aurantifolia) of in-vitro cultures were prepared by suppression subtractive hybridization (SSH).400 clones were isolated from cDNA library, nested PCR results show that the insert sizes ranged from 0.2kb to lkb,300 randomly selected colonies from the forward-subtractive library and of 100 colonies from the reverse-subtractive library were identified with the reverse Northern dot-blot screening method. All ESTs similarity were analysed by BLASTn and BLASTx. Sequencing results showed that167 ESTs represented different gene fragments,among which 115 genes are up-regulated expression and 52 genes are down-regulated expression by HLB infection.Among the 167 ESTs isolated and identified in Murraya paniculata. only 10ESTs had been reported on citrus in GenBank database, the others are mostly from Arabidopsis, Solanum and Ricinus communis. The function of 63 (about 36%) gene fragments was clearly unknown, while the rest 64% had function clarifying as disease defense, transcription,signal transduction, energy, metabolism and protein synthesis.The results indicated that the process of HLB-host interactions was complex and many genes could participate in the process. In this paper, we first reported the differentially expressed genes of Murraya paniculata induced byHLB infection, which established the foundation for understanding the molecular mechanisms and found disease-resistant genes.4 The RT-PCR results showed that the gene of miraculin-like protein, sthp2-21, ubiquitin-conjugating enzyme 8 were up regulated and chlorophyll A/B binding protein down regulated. Sequence analysis indicated that clonedSTH-21 gene consisted of 361 nucleotides (nt) containing 74 amino acids. Further comparison toSTH- 21 gene and Solanum tuberosum, Vitis vinifer showed that its identitywas 68% and 67%, respectively. Semi-quantitative RT-PCR revealed that expression of sth- 21 gene was induced by HLB, and the most abundantly time... |
PDF Full Text Request