Molecular diagnosis and direct quantification of cereal cyst nematode(Heterodera filipjevi) from field soil using TaqMan real-time PCR

Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying H.filipjevi from DNA extracts of soil.The primers and probe designed from the obtained RAPD-SCAR marker fragments of H.filipjevi showed high specificity to H.filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera spp.and 3 Pratylenchus spp.The qPCR assay is highly sensitive and provides improved H.filipjevi detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H.filipjevi in naturally infested field soils.There was a high correlation between the H.filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay.qPCR potentially provides a useful platform for the efficient detection and quantification of H.filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.

植物寄生线虫基因组学研究进展

植物寄生线虫是农作物主要病原物之一,每年造成作物大幅度减产和严重的经济损失。植物寄生线虫基因组学研究在揭示植物线虫与寄主互作分子机制和作物抗线虫品种的培育中发挥着不可替代的作用。随着高通量测序技术的广泛应用,多种重要的植物寄生线虫的基因组被破解和报道。在此基础上,通过比较基因组学进一步揭示了植物寄生线虫的起源和进化,功能基因组学的研究也取得了一系列重要的进展。本文对目前已报道的植物寄生线虫基因组的研究进展进行总结,在各基因组的基本特征、染色体组变异、串联重复序列、基因表达调控与基因协同表达、水平基因转移、效应蛋白发现和功能研究及基因家族扩张等方面进行综述,以期为植物线虫致病分子机制研究及防控新策略制定提供参考。

Evaluation of Chinese rice varieties resistant to the root-knot nematode Meloidogyne graminicola

The root-knot nematode Meloidogyne graminicola, which is distributed worldwide, is considered a major constraint on rice production in Asia. The present study used the root gall index and number of nematodes inside the roots to evaluate resistance/susceptibility to M. graminicola in different subpopulations of Oryza sativa(aus, hybrid aus, indica, hybrid indica, temperate japonica, tropical japonica). Nematode development in highly resistant varieties was also evaluated. Analyses of randomly selected 35 varieties showed the number of M. graminicola nematodes inside the roots correlated very strongly(r=0.87, P≤0.05) with the nematode gall index, and the results from pot and field experiments revealed similar rankings of the varieties for resistance/susceptibility. Among the 136 tested varieties, temperate japonica displayed the highest gall index, followed by tropical japonica, indica, hybrid indica, aus, and hybrid aus. Zhonghua 11(aus), Shenliangyou 1(hybrid aus) and Cliangyou 4418(hybrid indica) were highly resistant to M. graminicola under both pot and field conditions. Further examination of nematode development suggested that compared to susceptible rice, M. graminicola penetrated less often into highly resistant varieties and more frequently failed to develop into females. The promising varieties found in the present research might be useful for the breeding of hybrid rice in China and for the further development of practical nematode management measures.

Evaluation of the biocontrol potential of Aspergillus welwitschiae against the root-knot nematode Meloidogyne graminicola in rice(Oryza sativa L.)

The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas,and nematicides are neither ecofriendly nor cost effective.More acceptable biological agents are required for controlling this destructive pathogen.In this study,the biocontrol potential of Aspergillus welwitschiae AW2017 was investigated in laboratory and greenhouse experiments.The in vitro ovicidal and larvicidal activities of A.welwitschiae metabolites were tested on M.graminicola in laboratory experiments.The effect of A.welwitschiae on the attraction of M.graminicola to rice and the infection of rice by M.graminicola was evaluated in a greenhouse.The bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal action.The best larvicidal activity was observed at a concentration of 5×AW2017,which caused an 86.2%mortality rate at 48 h post inoculation.The highest ovicidal activity was recorded at a concentration of 5×AW2017,which resulted in an approximately 47.3%reduction in egg hatching after 8 d compared to the control.Under greenhouse conditions,the application of A.welwitschiae significantly reduced the root galls and nematodes in rice roots compared to the control.At a concentration of 5×AW2017,juveniles and root galls in rice roots at 14 d post inoculation(dpi)were reduced by 24.5 and 40.5%,respectively.In addition,the attraction of M.graminicola to rice roots was significantly decreased in the AW2017 treatment,and the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control plants.The results indicate that A.welwitschiae is a potential biological control agent against M.graminicola in rice.

Golden Promise barley(Hordeum vulgare) is a suitable candidate model host for investigation interaction with Heterodera avenae

Heterodera avenae(cereal cyst nematode, CCN) infects many cereal crops and causes serious yield losses worldwide. Interaction studies investigating H. avenae and its hosts are still in their infancy. In this study, a barley model plant, the Hordeum vulgare cultivar Golden Promise, was investigated for its potential as a candidate model host to study its interaction with H. avenae. CCN-infective juveniles were attracted by the root tips and gathered around the root elongation zones of Golden Promise on 0.7% water agar plates. The juveniles invaded the roots and developed successfully until maturation at 40 days after inoculation in sterile sand soil. The cryotomy and syncytium measurements indicated that the syncytia enlarged gradually throughout the development of the nematodes and caused the corresponding root regions to swell obviously. Quantitative real-time PCR analysis showed that the down-regulation of defence-related barley genes and up-regulation of development-related barley genes contribute to the understanding of compatible interaction between H. avenae and Golden Promise. Barley stripe mosaic virus(BSMV) virus-induced gene silencing(VIGS) can be used in the roots of Golden Promise. In conclusion, the Hordeum vulgare cultivar Golden Promise is a suitable candidate model host for interaction studies with Heterodera avenae. The studies presented above document the first CCN host that not only has published genome context but also be compatible to BSMV VIGS.

Identification of Heterodera schachtii on sugar beet in Xinjiang Uygur Autonomous Region of China

The sugar beet cyst nematode,Heterodera schachtii,is a major parasite of sugar beet which has been recognized and listed as a quarantine nematode in China and more than 20 countries and regions worldwide.A survey for important nematodes was undertaken in the sugar beet planting area of China during 2015-2018,and numerous cysts were collected from sugar beet fields in Xinyuan County,Xinjiang Uygur Autonomous Region of China.The observations of morphological and morphometric characteristics revealed that cysts,vulval cones and second-stage juveniles of the Xinjiang population were in the same range of each other and within those of other reported H.schachtii populations.Molecular analysis of rDNA-ITS,28S-D2/D3 and mtDNA cytochrome c oxidase subunit 1(COI)gene sequences suggested that the Xinjiang population clustered in a branch with those foreign populations,and the sequence similarity was as high as 99.81-100%.Moreover,this result was confirmed by PCR assay with species-specific primer SHF6 and rDNA2 of H.schachtii,and the pathogenicity test confirmed successful Xinjiang population reproduction in both plant hosts.In conclusion,based on morphological and molecular characterization,this study confirmed that the cyst nematode population collected from sugar beet fields in Xinjiang is H.schachtii.As far as we know,this is the first report of H.schachtii on sugar beets in Xinjiang,China.

基于五阶Bezier曲线的无人车避障轨迹规划

无人驾驶汽车的局部路径规划对于自动驾驶技术的推广有着至关重要的作用。为了研究无人驾驶汽车在运行过程中前方会出现会静止障碍物采用换道避障策略的情况,提出了基于五阶Bezier曲线的局部路径规划方法。首先,通过分析车辆性能极限及车辆碰撞边界确定换道过程中车辆的可行驶域,再进一步考虑车辆的物理特性提出轨迹曲线优化目标函数,确定五阶Bezier曲线的6个控制点,得到最优避障轨迹。然后利用CarSim和MATLAB/Simulink软件进行仿真实验验证。结果表明此方法能够规划出易于车辆跟踪的轨迹曲线,且针对不同车速情况下的换道避障能够分别产生此车速下的最优避障轨迹。

First report of cereal cyst nematode(Heterodera filipjevi) on winter wheat in Shandong Province, China

The cereal cyst nematodes(Heterodera avenae,Heterodera filipjevi,Heterodera latipons)are considered to be one of the most important plant parasitic nematodes attacking most cereals and can cause significant crop losses(Sikora 1988).In China,H.filipjevi(Madzhidov 1981)Stelter,1984,was first reported from Henan province(peng et al.2010)and a few years later in Anhui province and Xinjiang Uygur Autonomous Region(peng et al.2016,2018).

Assaying the potential of twenty-one legume plants in Medicago truncatula and M. sativa for candidate model plants for investigation the interactions with Heterodera glycines

Soybean cyst nematode Heterodera glycines is one of the most serious soil-borne pathogens in soybean production. However, the researches were limited in China due to lack of an effective pathosystem. In this study, we screened 21 legume Medicago plants in both Medicago truncatula and Medicago sativa to obtain candidate model plants for establishing a new pathosystem for legume-H. glycines interactions. The nematode infection of tested plants was assayed with Race 3 and 4 respectively, which were two dominant H. glycines inbred races in China soybean producing areas. The results showed that the model legume plant M. truncatula A17 failed to allow Race 3 of H. glycines to complete its life cycle, in contrast, it provided the Race 4 population to form several cyst nematodes, however, the female index(FI) value was approximately 1.6. Three M. sativa cultivars, including Xunlu, Aergangjin and Junren, provided either Race 3 or 4 of H. glycines to develop into mature cysts with their FI value below 5 as well. Our results demonstrated that legume plants in both M. truncatula and M. sativa were not likely to be a model plant for H. glycines because of an extreme high resistance.

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot,which results in a large quantity of annual yield loss in soybean production,by its secretions including Fusarium toxins and cell wall degrading enzymes,but not by the conidia themselves that do not underlie any virulence so far.Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone.We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis.Of them,except for one isolate that showed diversity of virulence to different soybeans(virulent to one soybean whereas avirulent to another soybean),the others were all virulent to the two tested soybeans:both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection,and their virulence had dosage effects that only conidia cultures of at least 5×10~6 conidia mL^(-1) could show virulence to soybean;however,the sole conidia of the F.solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted.Finally,we developed the specific cleaved amplified polymorphic sequences(CAPS)markers to easily differentiate Fusarium isolates.The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.

Potassium sulphate induces resistance of rice against the rootknot nematode Meloidogyne graminicola

Potassium(K),an important nutrient element,can improve the stress resistance/tolerance of crops.The application of K in resisting plant-parasitic nematodes shows that the K treatment can reduce the occurrence of nematode diseases and increase crop yield.However,data on K_(2)SO_(4)induced rice resistance against the root-knot nematode Meloidogyne graminicola are still lacking.In this work,K_(2)SO_(4)treatment reduced galls and nematodes in rice plants and delayed the development of nematodes.Rather than affecting the attractiveness of roots to nematodes and the morphological phenotype of giant cells at feeding sites,such an effect is achieved by rapidly priming hydrogen peroxide(H_(2)O_(2))accumulation and increasing callose deposition.Meanwhile,galls and nematodes in rice roots were more in the potassium channel OsAKT11 and transporter OsHAK5 gene-deficient plants than in wild-type,while the K_(2)SO_(4)-induced resistance showed weaker in the defective plants.In addition,during the process of nematode infection,the expression of jasmonic acid(JA)/ethylene(ET)/brassinolide(BR)signaling pathway-related genes and pathogenesis-related(PR)genes OsPR1 a/OsPR1 b was up-regulated in rice after K_(2)SO_(4)treatment.In conclusion,K_(2)SO_(4)induced rice resistance against M.graminicola.The mechanism of inducing resistance was to prime the basal defense and required the participation of the K^(+)channel and transporter in rice.These laid a foundation for further study on the mechanism of rice defense against nematodes and the rational use of potassium fertilizer on improving rice resistance against nematodes in the field.

A fragment of a 70-kDa Heterodera glycines heat shock protein(HgHSP70)interacts with soybean cyst nematode-resistant GmSHMT08

Soybean cyst nematode(SCN)Heterodera glycines is considered as the major constraint to soybean production.Gm SHMT08 at Rhg4 locus on chromosome 08,encoding a serine hydroxylmethyltransferase,is a major gene underlying resistance against H.glycines in Peking-type soybeans.However,the molecular mechanism underpinning this resistance is less well characterized,and whether Gm SHMT08 could interact with proteins in H.glycines remains unclear.In this study,yeast two-hybrid screening was conducted using Gm SHMT08 as a bait protein,and a fragment of a 70-kDa heat shock protein(Hg HSP70)was screened from H.glycines that exhibited interaction with Gm SHMT08.This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays.Our finding reveals Hg HSP70 could be applied as a potential candidate gene for further exploring the mechanism on Gm SHMT08-mediated resistance against SCN H.glycines.

Molecular and morphological characterization of stunt nematodes of wheat, maize, and rice in the savannahs of northern Nigeria

Stunt nematodes(Tylenchorhynchus spp.)are obligate migratory root ecto-parasitic nematodes found in the fields of many cultivated crops.These nematodes,with phyto-sanitary potential,are frequently ignored or misdiagnosed as pests,and this may pose a threat to food security.The accuracy of its identification based on a morphological approach has been challenged recently,due to the overlapping of the morphological and morphometric characters of the species.Consequently,the objective of this study is to identify and characterize stunt nematodes present in 54 fields cultivated with cereal crops(wheat,maize and rice)in the savannahs of northern Nigeria,using integrative taxonomy and molecular approaches.The molecular and morphological studies identified and confirmed the presence of T.annulatus as the occurring specie in the savannahs of northern Nigeria.The phylogenetic analysis was carried out using the internal transcribed spacer(ITS)and 28S genes of ribosomal DNA further confirmed the presence of T.annulatus.The first molecular characterization and sequences of the ITS and 28S rDNA gene for T.annulatus from Nigeria were provided by this research.Also,according to our literature search,this is the first report on T.annulatus in wheat,maize and rice in the savannahs of northern Nigeria.Further study to test the pathogenicity of the parasitic nematode species found in this survey is recommended for the prioritization and development of efficient management strategies.

Effect of Aspergillus niger NBC001 on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is one of the most important pests causing considerable damage to soybean(Glycine max(L.)Merr.)around the world.Biocontrol provides a strategy for sustainable nematode control.Previously,Aspergillus niger NBC001 was isolated from the cysts Heterodera spp.and able to control H.glycines and promote the growth of soybean in a pot experiment.In this study,the effects of NBC001 on H.glycines density and on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field were studied.The results showed that NBC001 could suppress H.glycines by 31.7%in the field.High-throughput sequencing analysis showed that NBC001 had no significant influence on soybean rhizosphere microbial community structure,indicating that seed coatdressing with the concentrated culture filtrate of NBC001 was safe for the soil ecological environment.In addition,high-throughput sequencing results demonstrated that at 10 days post transplantation,NBC001 increased the abundances of Actinobacteria and Acidobacteria,but decreased the abundances of Bacteroidetes and Gemmatimonadetes at the phylum level.Meanwhile,the abundances of Phyllobacterium,Ralstonia and H16 were increased,while the abundances of Adhaeribacter,Gemmatimonas,Sphingomonas,Flavisolibacter\Nere suppressed by application of NBC001.However,at 90 days post transplantation,NBC001 only increased the abundances oi Aeromicrobium and RB41 whereas it decreased the abundance of H16.The results indicated that application of NBC001 increased the relative abundances of the beneficial microorganisms such as Actinobacteria,Acidobacteria,Aeromicrobium and Phyllobacterium in the soil.In summary,NBC001 is an eco-friendly biocontrol agent for H.glycines control.

小麦非叶器官的光合特性及其对籽粒产量的贡献

以旗叶为对照,比较研究了小麦(Triticum aestivum)叶与非叶器官(穗、穗下节间、旗叶鞘)的光合生理以及对籽粒产量的贡献。结果表明:灌浆初期和中期,小麦穗下节间的叶绿素含量与旗叶差异不显著,但显著高于穗和旗叶鞘。灌浆后期,不同光合器官叶绿素含量由高到低依次为穗下节间、旗叶鞘、旗叶和穗。灌浆初期时叶片的群体光合速率占对照总群体光合的48.5%～50.0%,穗为29.5%～33.2%,穗下节间为12.2%～12.6%。灌浆中期,叶片的群体光合占比下降至42.9%～45.7%,穗提高至32.1%～34.4%。非叶光合器官中的RuBPCase和PEPCK活性在整个灌浆期都高于旗叶,且灌浆中期时穗部的PEPCK活性较灌浆初期时升高,而其他光合器官的PEPCK活性降低。穗下节间的ΦPSII显著大于旗叶,穗的Fv/Fm和ΦPSII均低于旗叶。穗部和叶片是籽粒产量的主要贡献者,其中,叶片的贡献为42.41%～44.97%,穗为17.77%～47.36%。研究结果将为小麦高产栽培的群体调控和品种选育提供理论依据。

小麦对小麦孢囊线虫抗病性研究进展

由小麦孢囊线虫引起的小麦孢囊线虫病发生分布范围广,防治困难,严重危害我国小麦生产。在我国危害小麦的孢囊线虫主要包括禾谷孢囊线虫(Heterodera avenae)和菲利普孢囊线虫(H.filipjevi)。种植抗病小麦品种是防治小麦孢囊线虫病最经济有效的措施,近10年来,我国科学家制定了小麦孢囊线虫抗性评价标准,测试了我国主推小麦品种和部分引进种质资源对小麦孢囊线虫的抗感性,鉴定到太空6号、新麦11、VP1620和Madsen等抗小麦孢囊线虫的优良材料,利用抗源创制了一系列的抗小麦孢囊线虫种质材料,从组织细胞学、基因组和转录组等解析了小麦抗孢囊线虫的机制。本文主要从小麦孢囊线虫致病型分化、抗性评价、抗性基因鉴定、抗性机制解析和抗病基因利用等方面进行综述。