Resistant Cucumber Cultivars Research Articles

Thiophanate-methyl and its major metabolite carbendazim weaken rhizobacteria-mediated defense responses in cucumbers against Fusarium wilt

AbstractThe effect of fungicides on the plant-rhizosphere microbiome is a subject of ongoing debate, but whether any alteration in the rhizosphere microbiome could affect plant health is an issue that has not been thoroughly investigated. To address this deficiency, we analyzed the rhizosphere microbiome of wilt disease—resistant and disease-susceptible cucumber cultivars to determine whether (and which) plant-associated microorganisms have a role in disease resistance. We further assessed whether the fungicides thiophanate-methyl and carbendazim affect the rhizosphere microbiome, which may contribute to the plant’s immune response. Based on results acquired with both radicle-inoculation and soil-inoculation methods, cultivars Longyuanxiuchun (LYXC) and Shuyan2 (SY2) were identified as being disease resistant, whereas Zhongnong6 (ZN6) and Zhongnong38 (ZN38) were susceptible. The microbiome structure differed substantially between the resistant and susceptible plants, with LYXC and SY2 each having a significantly greater Shannon index than Zhongnong38. These results revealed that the disease-resistant cucumber cultivars recruited more beneficial bacteria, i.e., Bacillus, in their rhizosphere soil; as such, Bacillus was identified as a keystone genus in the microbial co-occurrence network. Thus, the presence of Bacillus may help cucumbers defend against fungal pathogens within the rhizosphere. Bacillus subtilis strain LD15, which was isolated from LYXC rhizosphere soil, could suppress pathogen growth, in vitro, and reduce disease severity in pot assays. Moreover, evidence also confirmed the accumulation of LD1 in the rhizosphere soil of resistant cucumber cultivars. For LYXC, application of thiophanate-methyl or carbendazim altered the microbiome structure, decreased bacterial diversity, and reduced the abundance of Bacillus species. Finally, pot assays verified that fungicide application decreased the proportion of LD15 in rhizosphere soil. From a microbial perspective, thiophanate-methyl and carbendazim may weaken the rhizobacteria-mediated defense response of cucumbers against cucumber Fusarium wilt disease. Our findings reveal a role for the rhizosphere microbiome in protecting plants from pathogens and constitute a reference for assessing the ecotoxicological risk of pesticides to non-target soil microorganisms.

Knockout of elF4E using CRISPR/Cas9 for large-scale production of resistant cucumber cultivar against WMV, ZYMV, and PRSV.

CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.

CRISPR/Cas9 based mlo-mediated resistance against Podosphaera xanthii in cucumber (Cucumis sativus L.).

Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously mlo-based resistance was reported in various plants, including cucumber, with generated loss-of CsaMLO function mutants. However, mlo-based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the mlo-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for CsaMLO1, CsaMLO8, and CsaMLO11 of PM susceptible ADR27 cucumber inbred lines and CsaMLO mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect Podosphaera xanthii germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated CsaMLOs in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that CsaMLO8 mutation-based resistance was associated with the pre-invasive response, while CsaMLO1 and CsaMLO11 could be negative regulators in the post-invasive defense response in cucumber against P. xanthii. Although the loss-of CsaMLO8 function confers the highest penetration resistance, CsaMLO1 and CsaMLO11 double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with CsaMLO8 or post-invasive with CsaMLO1/CsaMLO11 mutations.

Genome-wide association study reveals candidate genes for gummy stem blight resistance in cucumber

Gummy stem blight (GSB), caused by Didymella bryoniae, is a serious fungal disease that leads to decline in cucumber yield and quality. The molecular mechanism of GSB resistance in cucumber remains unclear. Here, we investigated the GSB resistance of cucumber core germplasms from four geographic groups at the seedling and adult stages. A total of 9 SNPs related to GSB resistance at the seedling stage and 26 SNPs at the adult stage were identified, of which some are co-localized to previously mapped Quantitative trait loci (QTLs) for GSB resistance (gsb3.2/gsb3.3, gsb5.1, and gsb-s6.2). Based on haplotype analysis and expression levels after inoculation, four candidate genes were identified within the region identified by both Genome-wide association study (GWAS) and previous identified QTL mapping, including Csa3G129470 for gsb3.2/gsb3.3, Csa5G606820 and Csa5G606850 for gsb5.1, and Csa6G079730 for gsb-s6.2. The novel GSB resistant accessions, significant SNPs, and candidate genes facilitate the breeding of GSB resistant cucumber cultivars and provide a novel idea for understanding GSB resistance mechanism in cucumber.

Colletotrichum orbiculare strains distributed in Japan: race identification and evaluation of virulence to cucurbits.

The pathogen Colletotrichum orbiculare is causal fungus of cucurbit anthracnose. Multiple races have been identified in the United States, suggesting that it is necessary to cultivate suitable resistant cultivars and breed new cultivars with the most suitable resistance gene. This study examined the pathogenicity and virulence of 20 strains in Japan to clarify the existence of races and virulence differences. Based on the symptoms on inoculated cotyledons and true leaves of watermelon, we could evaluate the compatibility of each strain to each host cultivar. Our analysis based on the reaction to the host cultivar harboring the resistance gene Ar-1 (Cla001017) revealed the existence of three races in Japan. An alarming result was that a race that overcame Ar-1, which is a target gene in current watermelon breeding in Japan, is present in Japan. The cucumber and melon host cultivars showed diverse symptoms, whereas a squash cultivar was resistant to all strains. Three strains caused severe damage even to the most resistant cucumber cultivar 'Ban Kyuri' and resistant cultivars harboring Cssgr, a well-known gene conferring loss-of-susceptibility resistance. Screening genetic resources for novel resistance genes using strains with high virulence is of vital importance for watermelon, cucumber, and melon production.

Detection of Airborne Sporangia of Pseudoperonospora cubensis and P. humuli in Michigan Using Burkard Spore Traps Coupled to Quantitative PCR.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating foliar disease on cucumber resulting in reduced yields. In 2004, the pathogen re-emerged in the United States, infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. The pathogen cannot overwinter in Michigan fields but because of an influx of airborne sporangia CDM occurs annually. In Michigan, spore traps are used to monitor the presence of airborne P. cubensis sporangia in cucumber growing regions to guide the initiation of a fungicide program. However, Pseudoperonospora humuli sporangia, the causal agent of downy mildew on hop, are morphologically indistinguishable from P. cubensis sporangia. This morphological similarity reduces the ability to accurately detect P. cubensis from spore trap samples when examined with the aid of light microscopy. To improve P. cubensis detection, we adapted a qPCR-based assay to allow the differentiation between P. cubensis and P. humuli on Burkard spore trap samples collected in the field. Specifically, we evaluated the specificity and sensitivity of P. cubensis detection on Burkard spore trap tapes using a morphological-based and quantitative-PCR (qPCR)-based identification assay and determined whether sporangia of P. cubensis and P. humuli on Burkard samples could be distinguished using qPCR. We found that the qPCR assay was able to detect a single sporangium of each species on spore trap samples collected in the field with Cq values <35.5. The qPCR assay also allowed the detection of P. cubensis and P. humuli in samples containing sporangia from both species. However, the number of sporangia quantified using light microscopy explained only 54 and 10% of the variation in the Cq values of P. cubensis and P. humuli, respectively, suggesting a limited capacity of the qPCR assay for the absolute quantification of sporangia in field samples. After 2 years of monitoring using Burkard spore traps coupled with the qPCR in cucumber fields, P. humuli sporangia were detected more frequently than P. cubensis early in the growing season (May and June). P. cubensis sporangia were detected ∼5 to 10 days before CDM symptoms were first observed in cucumber fields during both years. This research describes an improved sporangial detection system that is key for the monitoring and management of P. cubensis in Michigan.

Cucumber mosaic virus resistance: Comparative proteomics of contrasting Cucumis sativus cultivars after long-term infection

Plant viruses are a significant threat to a wide range of host species, causing substantial losses in agriculture. Particularly, Cucumber mosaic virus (CMV) evokes severe symptoms, thus dramatically limiting yield. Activation of plant defense reactions is associated with changes in the cellular proteome to ensure virus resistance. Herein, we studied two cultivars of cucumber (Cucumis sativus) resistant host Heliana and susceptible host Vanda. Plant cotyledons were mechanically inoculated with CMV isolate PK1, and systemic leaves were harvested at 33 days post-inoculation. Proteome was profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility enhanced mass spectrometry. From 1516 reproducibly quantified proteins using a label-free approach, 133 were differentially abundant among cultivars or treatments by strict statistic and effect size criteria. Pigments and hydrogen peroxide measurements corroborated proteomic findings. Comparison of both cultivars in the uninfected state highlighted more abundant photosynthetic and development-related proteins in resistant cucumber cultivar. Long-term CMV infection caused worse preservation of energy processes and less robust translation in the susceptible cultivar. Contrary, compatible plants had numerous more abundant stress and defense-related proteins. We proposed promising targets for functional validation in transgenic lines: A step toward durable virus resistance in cucurbits and other crops. SignificanceSustainable production of crops requires an understanding of natural mechanisms of resistance/susceptibility to ubiquitous viral infections. We report original findings of comparative analysis of plant genotypes exposed to CMV. Deep discovery proteomics of resistant and susceptible cucumber cultivars, inoculated with widespread phytovirus, allowed to suggest several novel molecular targets for functional testing in plant protection strategies.

Long-term feeding on greenhouse cucumber affects life table parameters of two-spotted spider mite and its predator Phytoseiulus persimilis

Bio-ecological parameters of the two-spotted spider mite (TSSM), Tetranychus urticae Koch and its predators depend on the quality of its host plants. In this study, the life table parameters of TSSM and its main predator Phytoseiulus persimilis Athias-Henriot during different generations (1st, 5th, and 10th generations) were determined on the susceptible (‘Beth-Alpha’) and resistant (‘HED’) greenhouse cucumber cultivars. The results indicated that the longest total development time of TSSM was allocated to the first generation on the susceptible cultivar, but there was no significant difference among different generations on the resistant cultivar. The total development time of the 1st and 10th generations of P. persimilis on each of the susceptible and resistant cultivars had not significant difference. Total fecundity of TSSM was higher in the 10th generation on both cultivars tested than that of the former generations. However, the total fecundity of the predator drastically reduced after long-term rearing (10 generations) on both cultivars. The intrinsic rate of increase (r) of the 1st, 5th, and 10th generations of TSSM on the susceptible cultivar was 0.178, 0.238 and 0.319 day-1, respectively, and 0.157, 0.166 and 0.168 day-1 on the resistant cultivar, respectively which indicates the significantly negative effect of the resistant cultivar on this parameter. In P. persimilis, the value of the intrinsic rate of increase in the first and tenth generations on the susceptible cultivar was 0.275 and 0.267 day-1, respectively, and on the resistant cultivar was 0.244 and 0.210 day-1, respectively. By understanding these interactions in the long term, we will be able to develop suitable strategies for biological control of TSSM on cucumber.

Serial passage through resistant and susceptible cucumber cultivars affects the virulence of Fusarium oxysporum f. sp. cucumerinum.

Fusarium oxysporum f. sp. cucumerinum (Foc) is the causal pathogen of cucumber Fusarium wilt resulting in losses to cucumber production. To investigate the effects of the selective pressures of host plants on the virulence of Foc, a low virulence isolate, foc‐3b, was successively inoculated on resistant and susceptible cucumber cultivars for five generations. The virulence of the original isolate diverged; virulence was significantly strengthened after serial passage on the resistant cultivar and weakened on the susceptible plants (p ˂ .05). The expression of four virulence‐related genes of F. oxysporum, G‐protein α subunit gene fga1, sucrose nonfermenting 1 gene snf1, F‐box protein gene frp1, and Class V chitin synthase gene chsV, was quantified using real‐time PCR. All genes were significantly upregulated after serial passage on the resistant cultivar, compared to the original strain, and the expression of snf1 was downregulated in strains re‐isolated from the susceptible plants (p ˂ .05). A significant positive correlation was found between the expression levels of gene snf1, frp1, and chsV and disease severity of cucumber Fusarium wilt, suggesting these genes may impact virulence differentiation. This study will improve the management of cucumber Fusarium wilt and provide insight into the mechanisms underlying virulence of F. oxysporum.

Histological observation of cucumber infected with Corynespora cassiicola

In this study, we compared differences between cucumber cultivars D9320 (highly resistant) and D0401 (highly susceptible) after Corynespora cassiicola infection. There were no differences in conidial germination or penetration between these cultivars. The germ tube rapidly elongated within 24 h after inoculation and invaded the cucumber leaf tissue directly or via stomata 24 h after inoculation (hai). The hypersensitive response (HR) was observed using in vitro inoculation and the trypan blue staining method. Necrotic spots occurred only in the resistant cucumber cultivar D9320 in vitro inoculation. The water-soaked lesion appeared in D0401. The HR was observed earlier and stronger in D9320 than in D0401 in the light microscope. The phenomenon of hypersensitive cell death could be earlier observed by using light microscope. The accumulation of H2O2, lignin and callose was observed in D9320 and D0401 infected with C. cassiicola. The order of accumulation was H2O2, lignin, and then callose. H2O2, lignin and callose accumulation in D9320 occurred earlier and stronger than in D0401. The pattern of callose accumulation in D0401 was different from that of H2O2 and lignin. The intensity of the accumulation did not change over time in D0401. Consistent with lignin and callose accumulation, the expression levels of CsCALS and CsCCoAMT -increased earlier and were higher in the highly resistant D9320 cultivar than in the highly susceptible D0401 cultivar. The HR occurred after these accumulations. The amount of mycelium in D9320 was much lower than that in D0401 at 5 days post-infection (dpi). The HR plays a role in resisting infection of C. cassiicola.

오이 유묘와 과실을 활용한 검은별무늬병에 대한 저항성 품종 검정 방법

본 연구는 ê²€ì€ë³„ë¬´ëŠ¬ë³‘ê· ì˜ ë°°ì–‘ì  및 병원학 특성을 êµ¬ëª í•˜ê³ , 81개 시판 오이 í’ˆì¢ ì˜ ì €í•­ì„± 여부를 ê²€ì •í•˜ê¸° 위해 수행되었다. ì‹œí—˜ê· ì€ 2015ë „ 3월 말 경기도 평택에 위치한 시설 오이 재배농가에서 채취한 ë³‘ë“ ê³¼ì‹¤ë¡œë¶€í„° ë¶„ë¦¬í•˜ì˜€ê³ PDA상에서 배양하면서 í˜•íƒœì ì¸ 특성에 근거하여 ê²€ì€ë³„ë¬´ëŠ¬ë³‘ê· (C. cucumerinum PT1)으로 ë™ì •ë˜ì—ˆë‹¤. 상처 ìœ ë¬´ì™€ 과실 크기가 검은별무늬병의 발생에 미치는 영향을 ê·œëª í•˜ê¸° 위해 과일 크기를 ìœ ê³¼(10 cm 미만), 중과(10-20 cm), 대과(20 cm 초과)로 êµ¬ë¶„í•˜ê³ ê³¼ì‹¤ 크기별로 상처와 무상처 조건으로 오이 과실표면에 1×105 포자/ml 농도의 ë³‘ì›ê· ì„ ì ‘ì¢ í•˜ì˜€ë‹¤. 작은 과실에서는 표면 상처 여부에 관계없이 심한 수침상의 병반과 ë¹ ë¥¸ ë³‘ì›ê· ìƒìž¥ì„ 보이면서 피해 ì •ë„ê°€ 매우 컸던 반면, 중과와 대과에서는 상처 부위와 무상처 표면 모두에서 경미한 수침상의 병반이 나타났지만 식물체 안쪽 조직으로 병이 ì§„ì „ëœ 것은 상처 낸 과실에서 뿐이었다. 오이 ìœ ë¬˜ì™€ ìœ ê³¼ì— ê²€ì€ë³„ë¬´ëŠ¬ë³‘ê· ì˜ 포자 현탁액을 ì ‘ì¢ í•˜ì—¬ 81개 시판 오이 í’ˆì¢ ì˜ 병 ì €í•­ì„±ì„ 평가한 ê²°ê³¼, 미니오이와 피클오이 í’ˆì¢ êµ°ì€ 대부분 ì €í•­ì„±ì´ì—ˆë‹¤. 특히 ‘처음처럼’ í’ˆì¢ ì€ 아무런 병징이 나타나지 않았으며 나머지 í’ˆì¢ ë“¤ì€ ì €í•­ì„±-ì¤‘ë„ì €í•­ì„±ìœ¼ë¡œ 나타났다. 반면, 다른 í’ˆì¢ êµ°ì˜ 오이 í’ˆì¢ ì€ 대부분 감수성 í’ˆì¢ ìœ¼ë¡œ íŒëª ë˜ì—ˆë‹¤. C. cucumerinum PT1에 대한 오이의 병 ì €í•­ì„±ì€ í’ˆì¢ ê°„ ìœ ì˜í•˜ê²Œ 차이를 보였으며 일부 í’ˆì¢ ì— 있어서 ìœ ë¬˜ ê²€ì •ê³¼ ìœ ê³¼ ê²€ì • 간에 차이를 보였다. 따라서 검은별무늬병에 대한 ì €í•­ì„± í’ˆì¢ ì„ ì„ ë°œí•˜ê¸° 위해서 오이 ìœ ë¬˜ ê²€ì •ê³¼ ìœ ê³¼ ê²€ì •ì„ 병행하여 ì‚¬ìš©í• í•„ìš”ê°€ ìžˆë‹¤ê³ ìƒê°í•œë‹¤. 이상의 결과를 í† ëŒ€ë¡œ ì„ ë°œëœ ì €í•­ì„± í’ˆì¢ ì„ 농가에 ì ìš©í•œë‹¤ë©´ 오이 검은별무늬병 ë°©ì œë¥¼ 위한 ì‹¤ì§ˆì ì¸ 방법이 ë ìˆ˜ 있을 ê²ƒì´ë¼ê³ ìƒê°ëœë‹¤.

The expression patterns of Cucumis sativus WRKY (CsWRKY) family under the condition of inoculation with Phytophthora melonis in disease resistant and susceptible cucumber cultivars

Xu, X., Wang, R., Chao, J., Lin, Y., Jin, Q., He, X., Luo, S. and Wu, T. 2015. The expression patterns of Cucumis sativus WRKY (CsWRKY) family under the condition of inoculation with Phytophthora melonis in disease resistant and susceptible cucumber cultivars. Can. J. Plant Sci. 95: 1121–1131. Based on RNA-seq, we analyzed expression patterns of 55 CsWRKYs in the disease-resistant cultivar (JSH) and the disease-susceptible cucumber cultivar (B80) at 0 and 40 h post-inoculation with Phytophthora melonis. After inoculation with P. melonis, in JSH and B80, the number of up-regulated CsWRKY genes was both 25 with 22 commonly up-regulated genes, and there were 22 CsWRKY genes down-regulated in JSH and 23 in B80 with 18 commonly down-regulated genes, and there were eight CsWRKY genes with no or little change in transcript expression in JSH and seven in B80 with four common genes. Among the 55 CsWRKY genes, 17 genes showed significant differences (differences &gt; twofold) in the degree of change of gene transcription level between JSH and B80 from RNA-seq data analysis, and the results validated using real-time PCR showed that there were only six genes (CsWRKY2, 20, 26, 35, 44, 52) out of the 17 that exhibited significant differences (differences &gt; twofold) with four genes (CsWRKY2, 20, 26, 52) consistent with the data from RNA-seq and two genes (CsWRKY 35, 44) inconsistent with the data from RNA-seq. In addition, the six genes were strongly up-regulated after salicylic acid (SA) treatment with five genes (CsWRKY2, 20, 26, 35, 44) up to peak at 12 h and one gene (CsWRKY52) up to peak at 24 h in expression and that four CsWRKY genes (CsWRKY2, 20, 44, 52) out of six were strongly up-regulated with three genes (CsWRKY2, 20, 44) up to the highest point at 24 h and one gene (CsWRKY52) up after methyl jasmonate (MeJA) treatment. Based on the results above, we predicted CsWRKY2, 20, 26, 35, 44 and 52 may be involved in disease resistance of JSH against Phytophthora melonis by SA and (or) JA signaling pathway(s).

The expression patterns of Cucumis sativus WRKY (CsWRKY) family under the condition of inoculation with Phytophthora melonis in disease resistant and susceptible cucumber cultivars

Xu, X., Wang, R., Chao, J., Lin, Y., Jin, Q., He, X., Luo, S. and Wu, T. 2015. The expression patterns of Cucumis sativus WRKY (CsWRKY) family under the condition of inoculation with Phytophthora melonis in disease resistant and susceptible cucumber cultivars. Can. J. Plant Sci. 95: 1121-1131. Based on RNA-seq, we analyzed expression patterns of 55 CsWRKYs in the disease-resistant cultivar (JSH) and the disease-susceptible cucumber cultivar (B80) at 0 and 40 h post-inoculation with Phytophthora melonis. After inoculation with P. melonis, in JSH and B80, the number of up-regulated CsWRKY genes was both 25 with 22 commonly up-regulated genes, and there were 22 CsWRKY genes down-regulated in JSH and 23 in B80 with 18 commonly down-regulated genes, and there were eight CsWRKY genes with no or little change in transcript expression in JSH and seven in B80 with four common genes. Among the 55 CsWRKY genes, 17 genes showed significant differences (differences > twofold) in the degree of change of gene transcript...

Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthora melonis, Phytophthora capsici and water immersion in disease resistant and susceptible cucumber cultivars

L-type lectin receptor kinase (LecRK) proteins are an important family involved in diverse biological processes such as pollen development, senescence, wounding, salinity and especially in innate immunity in model plants such as Arabidopsis and tobacco. Till date, LecRK proteins or genes of cucumber have not been reported. In this study, a total of 25 LecRK genes were identified in the cucumber genome, unequally distributed across its seven chromosomes. According to similarity comparison of their encoded proteins, the Cucumis sativus LecRK (CsLecRK) genes were classified into six major clades (from Clade I to CladeVI). Expression of CsLecRK genes were tested using QRT-PCR method and the results showed that 25 CsLecRK genes exhibited different responses to abiotic (water immersion) and biotic (Phytophthora melonis and Phytophthora capsici inoculation) stresses, as well as that between disease resistant cultivar (JSH) and disease susceptible cultivar (B80). Among the 25 CsLecRK genes, we found CsLecRK6.1 was especially induced by P. melonis and P. capsici in JSH plants. All these results suggested that CsLecRK genes may play important roles in biotic and abiotic stresses.

Downy mildew resistant/susceptible cucumber germplasm (Cucumis sativus L.) genetic diversity assessment using ISSR markers

Downy mildew is one of the major foliar diseases of cucumber in which the new resistant cucumber cultivars are necessary to control this disease. Before improving the new cultivar, knowing the information about genetic diversity of cucumber germplasm is necessary. Thus, the objective of this study is to evaluate the genetic diversity of downy mildew resistant/susceptible cucumber germplasm. Forty cucumber accessions were screened for downy mildew disease reaction and were classified into five classes which the average of percent of symptomatic leaf area ranged from 20.1% (highly resistant) to 95.0% (highly susceptible). Then, these cucumber germplasms were analyzed with 20 inter simple sequence repeat (ISSR) markers. A total of 17 ISSR primers (85%) could amplify in all 40 cucumber accessions with the percent of polymorphic bands of each primer ranged from 55.56% to 100%. The polymorphism information content (PIC) ranged from 0.12 to 0.45 with the mean of 0.25, while the resolving power (RP) averaged at 1.82 and ranged from 0.50 to 3.20. The phylogenetic tree and principal component analysis revealed the association between downy mildew disease reaction and country of the origin of cucumber germplasm which could help the cucumber breeders to select the parental lines for improving the new cultivars that resist to downy mildew in the future.

Elevated expression of hydrolases, oxidase, and lyase in susceptible and resistant cucumber cultivars systemically induced with plant growth-promoting fungi against anthracnose

Susceptible and resistant cultivars of cucumber (cv. Suyo and cv. Shogoin fushinari, respectively) were evaluated for induction of systemic resistance by plant growth-promoting fungal isolates Phoma sp. GS8-1 and nonsporulating fungus GU21-2 against Colletotrichum orbiculare. Reduction in anthracnose disease was assessed at regular intervals of 0, 3, 6, and 9 days after challenge inoculation. Activities of endochitinase, β-1,3-endoglucanase, peroxidase (PO), polyphenol oxidase, and phenylalanine ammonia lyase (PAL) were assayed at the above intervals, while the activities of exochitinase and β-1,3-exogluconase were assayed at 9 days after challenge inoculation. The data showed that all the enzymes activities increased in plants treated with plant growth-promoting fungi (PGPF) compared with nontreated plants of both cultivars. The activities of endochitinase and β-1,3-endoglucanase were highly increased in PGPF-induced plants than in nontreated plants of both cultivars at different time points. Activities of PO, polyphenol oxidase, and PAL also increased between 3 and 6 days due to inoculation with PGPF and decreased thereafter. The activity of PAL at 9 days in PGPF-induced plants was at the same level as that of nontreated plants of both cultivars. Results of this investigation suggest that PGPF-induced systemic resistance is associated with not only high increases in the activities of chitinase and β-1,3-gluconases but also other plant defense-related enzymes such as PO, PPO, and PAL. Roles of other mechanisms rather than accumulation of defense-related enzymes in PGPF-mediated induced resistance in susceptible cultivars were demonstrated.

Development of Downy Mildew-resistant Cucumbers for Late-season Production in the Northeastern United States

Cucurbit downy mildew, a disease caused by the oomycete pathogen Pseudoperonospora cubensis (Berk. &amp; Curt.) Rostov., is a serious threat to cucumber (Cucumis sativus L.) production worldwide and can result in 100% yield losses in affected environments. In the last decade, strains of the pathogen have overcome the resistance of commercial cultivars in the United States, and currently no cultivar has robust resistance to the disease. This lack of resistance has been especially problematic for cucumber growers seeking to capture the late-season market, when downy mildew is ubiquitous throughout Eastern and Great Lakes production environments. Our objectives were to identify sources of resistance genes and to introgress these genes into high-quality, high-yielding breeding material. Using the moderately resistant cucumber cultivars Marketmore 97 and Ivory Queen as well as the Cornell-developed cultivars Platinum and Salt &amp; Pepper, we have developed lines with excellent disease resistance. In a trial of 27 lines that included Cornell breeding material and the most resistant cultivars and U.S. Department of Agriculture (USDA) accessions identified in previous studies, the Cornell breeding line DMR-NY264 had the highest level of downy mildew resistance and the highest yields under disease pressure. In New York, plants of DMR-NY 264 produced fruit until frost without fungicide application.

High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber

Using a high-resolution mapping approach, we identified a candidate gene for ZYMV resistance in cucumber. Our findings should assist the development of high-versatility molecular markers for MAS for ZYMV resistance. Zucchini yellow mosaic virus (ZYMV) causes significant disease, which leads to fruit yield loss in cucurbit crops. Since ZYMV resistance is often inherited recessively in cucumber, marker-assisted selection (MAS) is a useful tool for the development of resistant cucumber cultivars. Using 128 families of an F2:3 population derived from a cross between susceptible 'CS-PMR1' and resistant 'A192-18' cucumber inbred lines, we confirmed that ZYMV resistance is conferred by a single recessive locus: zym (A192-18) . We constructed a cucumber genetic linkage map that included 125 simple sequence repeat (SSR) markers segregating into 7 linkage groups (chromosomes). The zym (A192-18) locus was mapped to chromosome 6, at genetic distances of 0.9 and 1.3cM from two closely linked SSR markers. For high-resolution genetic mapping, we identified new molecular markers cosegregating with the zym (A192-18) locus; using cucumber genomic and molecular marker resources and screening an F2 population of 2,429 plants, we narrowed down the zym (A192-18) locus to a <50-kb genomic region flanked by two SSR markers, which included six candidate genes. Sequence analysis of the candidate genes' coding regions revealed that the vacuolar protein sorting-associated protein 4-like (VPS4-like) gene had two SNPs between the parental lines. Based on SNPs of the VPS-4-like gene, we developed zym (A192-18) -linked DNA markers and found that genotypes associated with these markers were correlated with the ZYMV resistance phenotype in 48 cucumber inbred lines. According to our data, the gene encoding VPS4-like protein is a candidate for the zym (A192-18) locus. These results may be valuable for MAS for ZYMV resistance in cucumber.

Identification and validation of powdery mildew (Podosphaera xanthii)-resistant loci in recombinant inbred lines of cucumber (Cucumis sativus L.)

Powdery mildew limits cucumber production worldwide. Most resistant cucumber cultivars become susceptible to powdery mildew at low temperatures. Resistance within a wide temperature range is therefore desirable for cucumber production. We constructed a cucumber genetic linkage map based on a population of 111 recombinant inbred lines derived from a cross between CS-PMR1, with strong and temperature-independent resistance, and Santou, with moderate and temperature-dependent resistance. The map spans 693.0 cM and consists of 296 markers segregating into seven linkage groups; the markers include 289 simple sequence repeats (SSRs), six sequence characterized amplified regions, and one inter simple sequence repeat. Due to the presence of 150 common SSR markers, we were able to compare our map with previously published maps obtained by using populations derived from inter- or intra-variety crosses. We also evaluated powdery mildew resistance of the recombinant inbred lines and identified seven quantitative trait loci (QTL) contributed by CS-PMR1 and two QTL contributed by Santou. Four QTL (pm3.1, pm5.1, pm5.2 and pm5.3) were successfully validated by using populations derived from residual heterozygous lines. Some of the QTL identified in our study are in good agreement with previously published results obtained with materials of different origin. The markers reported here would be useful for introducing high and temperature-independent resistance by accumulation of QTL from CS-PMR1 and Santou.

Cucumber vein yellowing virus isolate-specific expression of symptoms and viral RNA accumulation in susceptible and resistant cucumber cultivars

Since its outbreak in Spain during the fall of 2000, Cucumber vein yellowing virus (CVYV) has become into a limiting factor in cucurbit cultivation in this country, particularly in cucumber. The impact of the virus has been limited after the introduction of resistant cucumber cultivars and the implementation of cultural practices to control whitefly, the virus vector. In Spain, CVYV isolates are genetically similar and cucumber resistance breaking isolates have not been reported. A CVYV isolate from Jordan (Jor) was previously reported as being more virulent in cucumber cv. Beit Alfa than an isolate from Israel. To study the reactions of different cucumber cultivars to the virulent Jor isolate in comparison with a Spanish isolate of (Alm 1A), we have inoculated both isolates in 10 commercial cucumber cultivars with resistance to the virus. Also, we have used real-time RT-PCR to estimate virus accumulation of Jor and Alm 1A isolates in three of these cultivars, one described as susceptible and two as resistant to CVYV. The Spanish Alm 1A isolate caused mild or no symptoms in resistant cultivars whereas Jor isolate caused intense symptoms in all cultivars. Alm1 A genomic RNA accumulated similarly in the three cultivars, whereas the accumulation of Jor genomic RNA was lower in the resistant cultivars than in the susceptible cultivar. Also the Jor isolate and accumulated earlier and at a higher level than the Alm 1A isolate in the three cultivars.