Identification Of Pratylenchus From Rhizosphere Of Corn And Functional Study Of Two Genes Of P. Coffeae

Pratylenchus Filipjev(1936),also known as root-lesion nematodes,is a type of migratory plant parasitic nematode with a wide range of hosts.Its siblings,Heterodera spp.and Meloidogyne spp.,are the three most serious pathogenic nematodes on crops.They are small in size,infect plant roots,and have a certain degree of concealment,These characteristics often lead to the neglect of the harm it causes in agricultural production.Pratylenus coffeae can harm many kinds of food and cash crop,such as corn,soybean,tobacco,etc.,and pose a great threat to crop safety in China.At present,P.coffeae are widely distributed around the world,prevention and control have become a global challenge.Identifying and studying the pathogenic genes and their functions of P.coffeae is the latest breakthrough in screening safe and effective control targets.In order to clarify the types and distribution of root-lesion nematodes in corn planting areas in the Huanghuai region,the author isolated and identified Pretylenchus from 274 rhizosphere soil samples collected from 18 corn fields in 4 provinces of the Huanghuai region(Henan,Shandong,Jiangsu,and Anhui).A total of 5 short bodied nematode species were obtained,with a separation rate of 39 % for P.coffeae and 32 % for P.scribneri.The P.coffeae is a dominant species of corn root rot nematode in the Huanghuai region.To further understand the pathogenic mechanism of P.coffeae infection,this study investigated the functions of the Cathepsin B and L genes(Pc-CB and Pc-CL)specifically expressed in the esophageal gland of the nematode through various molecular biology techniques.We used q PCR technology to detect the expression characteristics of target genes in different insect states of P.coffeae,and explored their functions in the process of infection and pathogenicity of nematodes by inducing target gene silencing through in vitro RNAi;The subcellular localization of target proteins Pc-CB and Pc-CL in plants and their molecular mechanisms involved in regulating host defense responses were clarified using methods such as Agrobacterium tumefaciens mediation.The specific research results are as follows:1.Isolation,identification,and purification of nematodesThe improved Bellman funnel method was used to isolate root lesion nematodes from 274 maize rhizosphere soil samples collected from 18 cities in 4 provinces of the Huanghuai region,and their species were identified through a combination of morphological and molecular biology methods.The identification results showed that 172 out of 274 samples were isolated with a separation rate of 62.8 %,and a total of 5 species were isolated,including P.coffeae,P.scribneri,P.zeae,P.neglectus,and P.thornei.Among 172 root lesion nematode samples,the isolation rate of P.coffeae was 39.5 %,the isolation rate P.scribneri was 32.6 %,and the isolation rates of P.zeae,P.neglectus and P.thornei were 12.8 %,5.8 %,and 9.3 %,respectively.P.coffeae is the dominant species of root lesion nematodes in the Huanghuai region.After purification and cultivation,26 purified populations were obtained.Research has found that P.coffeae and P.scribneri have been isolated from 18 cities,among which Xuzhou City in Jiangsu Province and Jinan City in Shandong Province have the phenomenon of composite infection of these two types of nematodes;The invasion and damage of P.zeae were first discovered in the rhizosphere of maize in Henan and Jiangsu provinces,and this nematode is a new recorded species in Henan and Jiangsu provinces.The first discovery of P.coffeae infecting and harming corn in Jiangsu and Anhui provinces,the first discovery of P.scribneri and P.neglectus infecting and harming corn in Anhui province,and the first discovery of P.thornei infecting and harming corn in Shandong province.2.Sequence analysis of the Pc-CB and Pc-CL genes of P.coffeaeThe research team obtained the full length transcriptome data of P.coffeae by high-throughput sequencing,and obtained the full length sequence of the Pc-CB gene 3370 bp by PCR amplification.The ORF length is 1116 bp,encoding 337 aa.The DNA coding region contains six introns,the theoretical molecular weight of the protein(MW)is 41.68 k Da,and the equipotential point p I is 4.50.The total length of the Pc-CL gene is 2013 bp,the ORF length is 1269 bp,encoding 422 aa,the DNA coding region contains 12 introns,the theoretical molecular weight(MW)of the protein is 48.30 k Da,and the equipotential point p I is 4.50.3.Expression characteristics and tissue localization of Pc-CB and Pc-CL genes in P.coffeaeq RT-PCR was used to detect the transcriptional differential levels of Pc-CB and Pc-CL in the four stages of P.coffeae(female,male,larva,and egg).It was found that Pc-CB and Pc-CL were expressed in all four stages of P.coffeae,with the highest expression levels in females and the lowest expression levels in males.Using nematode c DNA as a template,in vitro synthesis of digoxin labeled Pc-CB and Pc-CL antisense and antisense m RNA probes was carried out.In situ hybridization technology was used to confirm the specific expression of Pc-CB and Pc-CL in the esophageal gland cells of P.coffeae.4.Functional study of Pc-CB and Pc-CL in the infection and pathogenesis of P.coffeaeUsing in in vitro RNAi to silence target genes,within a certain time range,the silencing efficiency of the target gene continuously increases with the prolongation of its ds RNA soaking time.After 36 hours of soaking,the expression levels of PcCB and Pc-CL were the lowest in P.coffeae.Compared to the foreign gene e GFP silencing for 36 hours,the expression of Pc-CB decreased by 63.6 % and the expression of Pc-CL decreased by 64.3 %.The reproduction rate of P.coffeae treated with Pc-CB and Pc-CL ds RNA for 36 hours on carrot callus decreased by 47.7 % and 51.4 %,respectively,compared to the water control group,with significant differences(P < 0.05).Inoculate P.coffeae from different treatment groups onto the roots of maize seedlings with consistent growth.After 60 days,the results showed that the infectivity and pathogenicity of P.coffeae to host corn were significantly reduced in the Pc-CB and Pc-CL RNAi 36 h groups(P < 0.05).Inoculate different treatment groups of P.coffeae on the roots of corn seedlings for 72 h.The fuchsin staining results showed that the number of nematodes in the corn roots of the Pc-CB and Pc-CL RNAi 36 h treatment groups decreased by 64.3% and 58.3%,respectively,compared to the control group,with significant differences(P < 0.05).This indicates that after Pc-CB and Pc-CL silencing,the infectivity of the nematode to the host is significantly reduced.The above results indicate that Pc-CB and Pc-CL play important roles in the reproduction,infection,and pathogenesis of P.coffeae.5.Subcellular localization and Western blot detection of P.coffeae Pc-CB and Pc-CL in N.benthamianaUsing Agrobacterium mediated transient expression method,the recombinant protein Pc-CB labeled with GFP was identified to be located in the cytoplasm and nucleus of N.benthamiana,while Pc-CL was located in the cytoplasm of N.benthamiana.The Western blot validation results showed that the recombinant proteins PEG104-Pc-CB and PEG104-Pc-CL were successfully expressed on N.benthamiana.6.Effects of Pc-CB and Pc-CL in P.coffeae on plant immunityUnder various abiotic stress,plants can accumulate excess H2O2 in their bodies,leading to the outbreak of reactive oxygen species(ROS),which is also an important indicator of the successful recognition of pathogens and pathogen related pattern molecules(PAMPs)in plants.Using Agrobacterium mediated transient expression of PEG104-Pc-CB and PEG104-Pc-CL in tobacco,it was found that the accumulation of reactive oxygen species significantly decreased under the stimulation of bacterial flagella protein flag22.At the same time,q PCR analysis found that the transient expression of Pc-CB in N.benthamiana.disease progression related genes only showed insignificant downregulation of Pti5,while other PTI related genes and pathway genes were significantly downregulated(P < 0.05);Transient expression of Pc-CL,tobacco salicylic acid pathway(PAL)and jasmonic acid pathway(LOX)genes were significantly inhibited(P < 0.05),while the expression of PR1 and PTI related genes Pti5 of jasmonic acid pathway were not significantly down regulated,and other PTI related genes were significantly down regulated(P < 0.05).The RBP-1/GAP2 elicitor and the recombinant protein were injected into healthy tobacco leaves,and it was found that the effector proteins PcCB and Pc-CL could specifically inhibit necrosis of tobacco leaves.This indicates that Pc-CB and Pc-CL proteins can inhibit the host’s PTI and ETI reactions.