两种植物病原黄单胞菌基因组中同义密码子使用的分析

 根据已释放的基因组序列,对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv. campestris,Xcc)和地毯草黄单胞菌柑桔致病变种(X. axonopodis pv. citri,Xac)的密码子使用进行了分析。相对同义密码子使用值(relative synonymous codon usage,RSCU)的计算表明,它们具有高度相似的密码子使用模式。2个基因组密码子第3位的GC含量(GC3s)平均达0.806±0.077(Xcc)和0.791±0.075(Xac),倾向于使用GC含量较高的密码子。对有效密码子数量和密码子适应指数的分析表明,Xcc与Xac基因组中,高表达基因具有较高的GC含量,倾向于使用少数种类的密码子,而低表达基因具有较高的AT含量,倾向于随机地使用密码子。对密码子使用绝对次数进行的对应分析也证明了上述结论。同时,计算也证明了基因在基因组中的位置不影响密码子使用的模式。因此,基因组的GC含量、基因的表达水平和基因的种类与起源是影响这2个基因组密码子使用的主要因素。     

Molecular characterization of the incitant of cowpea bacterial blight and pustule, Xanthomonas campestris pv. vignicola

Strains of Xanthomonas campestris pv. vignicola (Xcv), isolated from cowpea leaves with blight or minute pustules and collected from various geographic areas, were selected on the basis of pathological and physiological features. All strains were analyzed for genotypic markers by two methods: ribotyping with EcoRI endonuclease, and RFLP analysis with a plasmid probe (pthB) containing a gene required for pathogenicity from Xanthomonas campestris pv. manihotis. Ribotyping revealed a unique pattern for all the strains that corresponded to the previously described ribotype rRNA7. Based on polymorphism detected by pthB among Xcv strains, nine haplotypes were defined. The observed genetic variation was independent of the geographic origin of the strains and of pathogenic variation. Some haplotypes were widely distributed, whereas others were localized. In some cases, we could differentiate strains isolated from blight symptoms and pustules according to haplotypic composition. However, in most cases, no significant differences were observed. Our results and the previous pathogenic and biochemical characterizations suggest that the strains isolated from leaves with blight symptoms or minute pustules belong to the same pathovar. We provide information on pathogen diversity that can be used to identify and characterize resistant germplasm.     

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow-pigmented bacteria isolated from blight-affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re-emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.     

The Effect of Irrigation Practices on the Spatio-temporal Increase of Asiatic Citrus Canker in Simulated Nursery Plots in Reunion Island

Asiatic citrus canker is a potentially severe disease of several citrus species and cultivars in many tropical and subtropical areas. In such areas, infected nursery plants constitute an important source of primary inoculum for newly established citrus groves. The influence of overhead, drip, and mist irrigation systems on the development of Asiatic citrus canker was studied in simulated, Mexican-lime nurseries in Reunion Island. Overhead irrigation exacerbated the increase of disease incidence and severity caused by a streptomycin-resistant strain of Xanthomonas axonopodis pv. citri. The temporal development of Asiatic citrus canker for overhead irrigated nursery plots was best described by an exponential model, because disease incidence in these plots did not come close to an asymptote during the experimental period. This can be explained by the continuous production of new growth, susceptible to infection by Xanthomonas axonopodis pv. citri, and splash dispersal of Xanthomonas axonopodis pv. citri associated with overhead irrigation. Based on spatial correlation and spatio-temporal analyses, aggregated disease patterns were found irrespective of the irrigation system. In overhead-irrigated plots, the spread of Xanthomonas axonopodis pv. citri lacked directionnality. Rainstorms of short duration and high intensity were apparently associated with disease increase in drip-irrigated plots. There is a need to improve cultivation practices in Reunion Island citrus nurseries to minimize Asiatic citrus canker incidence in nurseries and to minimize the introduction of Xanthomonas axonopodis pv. citri to new groves.     

Effect of temperature and leaf wetness duration on infection of sweet oranges by Asiatic citrus canker

Asiatic citrus canker, caused by Xanthomonas smithii ssp. citri , formerly X. axonopodis pv. citri , is one of the most serious phytosanitary problems in Brazilian citrus crops. Experiments were conducted under controlled conditions to assess the influence of temperature and leaf wetness duration on infection and subsequent symptom development of citrus canker in sweet orange cvs Hamlin, Natal, Pera and Valencia. The quantified variables were incubation period, disease incidence, disease severity, mean lesion density and mean lesion size at temperatures of 12, 15, 20, 25, 30, 35, 40 and 42°C, and leaf wetness durations of 0, 4, 8, 12, 16, 20 and 24 h. Symptoms did not develop at 42°C. A generalized beta function showed a good fit to the temperature data, severity being highest in the range 30–35°C. The relationship between citrus canker severity and leaf wetness duration was explained by a monomolecular model, with the greatest severity occurring at 24 h of leaf wetness, with 4 h of wetness being the minimum duration sufficient to cause 100% incidence at optimal temperatures of 25–35°C. Mean lesion density behaved similarly to disease severity in relation to temperature variation and leaf wetness duration. A combined monomolecular-beta generalized model fitted disease severity, mean lesion density or lesion size as a function of both temperature and duration of leaf wetness. The estimated minimum and maximum temperatures for the occurrence of disease were 12°C and 40°C, respectively.     

Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field

A total of 404 isolates of Xanthomonas campestris pv. vesicatoria , obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2 , shared taxonomic group B strain characteristics, including the presence of the β-protein band, positive amylolytic activity and inability to oxidize cis -aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot-susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.     

Local and regional spread of banana xanthomonas wilt (BXW) in space and time in Kagera,Tanzania

Banana is an important crop in the Kagera region of Tanzania. Banana xanthomonas wilt (BXW) was first reported in Kagera in 2006, and is now an important limiting factor in banana production, because all cultivars are susceptible and infected plants can fail to produce fruit. BXW is caused by Xanthomonas campestris pv. musacearum (Xcm), which is spread by farm tools, infected planting materials, and pollinating insects. Practices that address Xcm dissemination, such as mat removal, debudding and tool sterilization, have not prevented the spread of BXW in the region. Disease surveys were conducted in Kagera from 2007 to 2011 to assess BXW presence, monitor its intensity and evaluate its socioeconomic impacts. Spatiotemporal clusters of BXW were analysed with Arc GIS and sas . The relationship between BXW clusters and environmental variables was examined using bivariate correlations in spss ; two modelling approaches, MaxEnt (maximum entropy) and logistic regression, were used to predict the potential distribution of BXW in Tanzania. Disease progress over time was best described with the Gompertz model. Significant clustering of BXW was observed in all years and hotspots were located in the Muleba, Karagwe, Misenyi and Bukoba rural districts. These findings suggest that BXW spreads rapidly over short distances. BXW clusters were positively correlated with rainfall and negatively with temperature and altitude. According to MaxEnt, precipitation was the main factor associated with BXW development. MaxEnt and logistic regression predicted a wide potential distribution of BXW in Tanzania because the climate in all banana‐growing regions is conducive for its establishment.     

安徽省水稻品种对水稻白叶枯病的抗性及白叶枯病小种鉴定

为明确安徽省白叶枯病菌小种组成及常用、备用品种对该病的抗性,用白叶枯病强毒性小种FuJ和YN24、中等致病力的安徽省优势小种AH以及弱致病小种YN7对安徽省常用及备用水稻品种进行人工接种鉴定;用鉴别品种IRBB5、IRBB13、IRBB3、IRBB14、IRBB2、R24对安徽的白叶枯病菌株进行鉴定.结果表明,有3.5％的品种抗FuJ,15.4％的品种抗YN24,29.8％的品种抗AH;安徽省白叶枯病菌小种有R2、R5和R8,其中R5为优势小种.抗AH的品种可以用于安徽的水稻生产;生产中应防止FuJ和YN24等毒性强的菌株传入.     

A study on the molecular mechanism of resistance to amicarthiazol in Xanthomonas campestris pv. citri

Three amicarthiazol-resistant mutants (Xuv10, Xuv20 and Xuv40) were obtained by UV induction and used in this study. Minimal inhibition concentrations (MICs) of amicarthiazol against the growth of mutants and wild-type isolate were 400 and 100 microg ml(-1) respectively. Inhibition by amicarthiazol of succinate dehydrogenase (SDH) activities of Xanthomonas campestris pv. citri (Hasse) Dye wild-type isolate (Xcc) and three resistant mutants derived from this isolate were assayed using triphenyltetrazolium chloride (TTC). The SDH activities of these mutants were significantly lower than that of Xcc. The complete nucleotide sequences of four subunits (SdhA, SdhB, SdhC and SdhD) of succinate-ubiquinone oxidoreductase (SQR) were cloned by polymerase chain reaction (PCR) amplification. An amino acid mutation (His229--> Leu229) in sdhB was found to confer resistance of X. campestris pv. citri to amicarthiazol. It is suggested that this mutation alters the SDH complex in some way that prevents binding of amicarthiazol.     

Effect of synthetic and organic soil fertility amendments on southern blight, soil microbial communities, and yield of processing tomatoes

ABSTRACT Soil fertility amendments, including composted cotton-gin trash, swine manure, a rye-vetch green manure, or synthetic fertilizers, were applied to subplots and tillage on bare soil; or tillage followed by surface mulch with wheat straw were applied to main plots to determine the effect on the incidence of southern blight caused by Sclerotium rolfsii, yield of processing tomato, and soil microbial communities. The amendment-tillage interaction was significant in 1997 and disease incidence was 67% in tilled bare soil receiving synthetic fertilizers; whereas disease incidence was 3, 12, and 16% in surface-mulched plots amended with a composted cotton-gin trash, swine manure, or a rye-vetch green manure. The amendment effect was significant in 1998, and disease incidence was 61% in plots receiving synthetic fertilizer and was 23, 44, and 53% in plots receiving cotton-gin trash, swine manure, or rye-vetch green manure, respectively. In 1997, yields were highest in tilled surface-mulched plots amended with synthetic fertilizers, cotton-gin trash, or swine manure, respectively. In 1998, yields were low in all plots and there were no significant differences in yield due to treatment. Propagule densities of antagonistic soil fungi in the genus Trichoderma were highest in soils amended with composted cotton-gin trash or swine manure in both years. Propagule densities of fluorescent pseudomonads in soil were higher in plots amended with organic amendments than with synthetic fertilizers in both years. Propagules densities of enteric bacteria were elevated in soils amended with raw swine manure biosolids in both years. Our research indicates that some organic amendments, such as cotton-gin trash, reduced the incidence of southern blight in processing tomato and also enhanced populations of beneficial soil microbes.     

Functional analysis of walnut polyphenol oxidase gene (JrPPO1) in transgenic tobacco plants and PPO induction in response to walnut bacterial blight

Walnut (Juglans regia) is economically important for both its wood and nut nutritional value, but it is susceptible to diseases such as walnut bacterial blight, caused by Xanthomonas arboricola pv. juglandis (Xaj). Walnuts contain many phenolic compounds, providing a good model on which to study polyphenol oxidase (PPO). We inoculated the detached walnut fruits of cultivars Ford, Chandler, Franquette, Robert Livermore, and Payne with Xaj and measured the induction of PPO activity in infected sites and adjacent to infected sites. Compared to infected and uninfected sites, PPO activity was induced significantly in areas adjacent to infected sites in all cultivars except Ford. Ford and Franquette, presenting the lowest and highest PPO activity, showed the largest and smallest mean diameter spots in response to Xaj, respectively. Polyacrylamide gel electrophoresis confirmed monophenol oxidase activity of walnut PPO in the assessed tissues. Then, we revealed the antipathogenic potential of walnut PPO through Agrobacterium tumefaciens-mediated walnut JrPPO1 gene transfer into tobacco (Nicotiana tabacum). Two transformed tobacco lines overexpressing the JrPPO1 gene were regenerated successfully and challenged with Pseudomonas syringae pv. tabaci. Transgenic lines showed significantly higher PPO activity and lower disease severity to the pathogen compared to the control. However, a significant difference in disease severity and PPO activity level was observed between the two transgenic lines. Our results demonstrate a potential defence-related role of PPO in transgenic tobacco and its induction in areas adjacent to infection sites in walnut cultivars treated with Xaj.     

辣椒细菌性疮痂病病原菌分类、检测及综合防治研究进展

辣椒细菌性疮痂病是一种世界性分布的细菌性病害,该病能引起辣椒严重的产量损失和品质下降。国外特别是美国对该病害研究较早且较深入,国内相关研究几乎是空白。本文主要围绕病原菌的分类、检测和病害综合防治等研究进展做一概述。     

The genetic characterization of Xanthomonas arboricola pv. juglandis,the causal agent of walnut blight in Poland

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC‐, BOX‐PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I‐391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.     

Characterization,genetic diversity and distribution of Xanthomonas campestris pv. campestris races causing black rot disease in cruciferous crops of India

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Effect of weather conditions on local spread and infection by pea bacterial blight (Pseudomonas syringae pv. pisi)

The effect of weather conditions on simultaneous local (plant to plant) spread and infection of peas (Pisum sativum) with bacterial blight (Pseudomonas syringae pv. pisi) was investigated by exposing susceptible bait plants for 24 h periods in infected field plots. Following exposure, bait plants were maintained in a glasshouse. Disease symptoms were recorded on 55 out of a total of 105 days on which plants were exposed. Nearly all of these infection events (53) were associated with the occurrence of rain. A series of Generalised Linear Models was fitted to the data to examine the relationships of the mean number of lesions (m) or the proportion of bait plants infected (p) to various weather variables and disease levels in the plots. Rainfall rate and wind run were the most important explanatory variables for the mean number of lesions followed by maximum temperature, rainfall duration, rainfall in the previous week and disease incidence in the surrounding crop. However, rainfall duration and disease incidence were the most important for the proportion of bait plants infected, followed by wind run. A four variable model relating the mean number of lesions to the rainfall rate, wind run, maximum temperature and either rainfall the previous week or disease incidence in the surrounding crop was considered to be the most useful for use in simulation studies.     

Effects of strip intercropping of potatoes with non‐hosts on late blight severity and tuber yield in organic production

The effects of strip cropping of potatoes with cereals or a grass‐clover mix in and perpendicular to the main wind direction on foliar late blight severity and tuber yield were studied in large‐scale field experiments in Germany. Disease progress was assessed in 12–18 sections per plot and yields determined from the same sections. In 2000, plot size was 3 × 10 m and there were no disease reductions apparently due to interplot interference. In 2001 and 2002, with plot sizes of 6 × 18 and 6 × 36 m in strip‐cropped potatoes, disease was significantly reduced by 9–20% and 4–12%, respectively, compared to pure stands of potato, with the greatest reductions in plots planted perpendicular to the wind and neighboured by grass‐clover. The most important factors contributing to disease reduction were loss of inoculum outside of the plots and barrier effects of neighbouring non‐potato hosts. Only 0–20% of the overall yield variation could be explained by the area under the disease progress curve, depending on cultivar and year. In one year disease effects on the yield of a moderately resistant cultivar were higher than on the yield of a susceptible cultivar. This was probably caused by differences in bulking behaviour. Nutrient limitation appeared to be more important than disease in reducing yields. Yields in the edge potato rows directly neighboured by cereals were significantly reduced in all 3 years, but competition by cereals did not change the disease–yield‐loss relationship. Strip intercropping might be a useful component in an overall management strategy to reduce incoming late blight inoculum.     

桃细菌性穿孔病及防治研究进展

树生黄单胞菌李致病变种Xanthomonas arboricola pv.pruni(Xap)是一种危害桃树的植物病原细菌,会导致桃树发生细菌性穿孔病。本文主要对该细菌性穿孔病菌的分布、命名、遗传多样性、致病机理和防治技术的研究进展进行了总结,重点阐述了桃细菌性穿孔病的不同防治方法,以期能提供新的防治思路,提升防治水平,保障桃果的安全生产。     

低氮环境下燕麦与箭豌豆间混作与刈割时间对饲草产量及质量特性的影响

对北京市延庆县旱作低N土壤环境下燕麦与箭豌豆间混播6种方式与比例对饲草产量与质量特性影响进行了研究。结果表明,在低N环境下,与燕麦单作相比,箭豌豆与燕麦间混播不仅可增加饲草中干物质产量,饲草N产量也可增加30%~130%;而且燕麦与箭豌豆间混作对饲草质量也有显著影响(P<0.01)。间混作能够降低饲草中ADF与NDF含量,提高燕麦开花后饲草RFV值。4种间混作处理在饲草产量与饲草品质方面各有优点,也各有不足。在低N环境下,如果以饲草产量和饲草N产量为主要追求目标,则燕麦与箭豌豆1∶1邻行间作方式为最佳播种模式,其次依次分别是燕麦与箭豌豆1∶1同行混播、燕麦与箭豌豆1∶2同行混播、燕麦与箭豌豆2∶1同行混播。燕麦与箭豌豆间混播最佳刈割期为燕麦乳熟期 箭豌豆满荚期,因为此时混合饲草产量最高,饲草N产量与RFV较高,而ADF与NDF较低。     

Effect of primary inoculum sources of bean common bacterial blight on early epidemics,seed yield and quality aspects

Rainfed wetland rice (RWR) had more species in common with irrigated wetland than dryland rice agroecosystems. Across ecosystems, higher pest densities and losses were recorded in RWR sites. We hypothesise that under low pressure from natural enemies, vegetative stage losses became particularly high due the combination of whorl maggot and caseworm damage combined with the physiological stress of transplanting shock. Both of these pest groups benefited from an expanded vegetative period common in RWR agroecosystems. Losses in older rice were probably due to stemborers. RWR is more prone to an array of physiological stresses than irrigated rice that we believe minimises crop compensation to accentuate insect losses. Chemical control is uneconomical mainly due to the low yield potential of RWR and the poor efficacy of applied insecticide.