Soil‐less bioassays for early screening for resistance to imazapyr in sunflower (Helianthus annuus L.)

BACKGROUND: Rapid and efficient diagnostic tests for early screening of herbicide resistance are convenient alternatives to field screening methods. There is a need for a quick, reliable and cost‐effective method for rapid diagnosis of imidazolinone resistance in sunflower (Helianthus annuus L.). RESULTS: Two seed germination bioassays were developed. Seeds from three sunflower inbred lines differing in resistance to imidazolinones were germinated either on solid culture medium or placed in plastic pots filled with commercial perlite. After 8 days incubation under controlled conditions, both assays successfully distinguished susceptible genotype from the resistant and intermediate ones. The susceptible genotype showed arrested root growth at all herbicide treatments (root length < 1 cm). The resistant genotype developed a complete root system even when exposed to the highest dose of herbicide. However, no definite differences were observed for the intermediate and resistant genotypes with respect to root growth under the different herbicide treatments. CONCLUSION: The simple and rapid screening assays described in the present study were useful in discriminating imidazolinone resistance at the seedling stage. Therefore, these bioassays could be potential tools for early screening of imidazolinone resistance genes from large sunflower populations. Copyright © 2009 Society of Chemical Industry     

A Laboratory Assay for Phytophthora infestans Resistance in Various Solanum Species Reflects the Field Situation

Physiological and molecular research on resistance responses of Solanum tuberosum cultivars and partially resistant Solanum species to Phytophthora infestans requires a reliable resistance test that can be used in the laboratory. Laboratory tests performed on detached leaves and intact plants were compared with field tests for similarity of late blight reactions. Detached leaves from field-grown plants were as resistant as detached leaves from climate chamber-grown plants when challenged with P. infestans. However, detached leaves incubated in covered trays at high relative humidity were more susceptible than detached leaves kept in open trays or leaves on intact plants. The incubation conditions of detached leaves in covered trays rather than detachment itself appeared to affect the resistance expression. Detached leaves of some wild Solanum genotypes became partially infected, whereas intact plants were completely resistant when inoculated. Inoculation of leaves on intact plants, however, resulted in lower infection efficiencies. These limitations should be taken into account when choosing the appropriate inoculation method for specific purposes. For resistance screening, laboratory tests proved to be a good alternative for field tests. The ranking of resistance levels for twenty plant genotypes was similar under laboratory and field conditions.     

Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae , the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae . A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae -resistant accessions of the wild sea beet ( Beta vulgaris ssp. maritima ), which might be used to restrict the transmission of BNYVV, were identified.     

Factors affecting expression of resistance in pepper (Capsicum annuum) to blight caused by Phytophthora capsici in screening trials

The effects of plant age, fungus isolate, zoospore concentration and inoculation method on the resistance of pepper ( Capsicum annuum) to blight ( Phytophthora capsici) were studied under controlled conditions. Each of the factors studied produced differing responses in the pepper lines tested hut line CNPH 286 proved to be a reliable source of resistance and is now being used in a breeding programme. An inoculation method for screening pepper lines for blight resistance is proposed, using a standardized zoospore suspension applied to the base of plants 45 days from sowing.     

A screening test for Mycosphaerella brassicicola on Brassica oleracea

A greenhouse screening method for resistance to ringspot (Mycosphaerella brassicicola) inBrassica oleracea is described. High infection levels were achieved by spraying young plants by mycelial inoculum enriched with 3% sucrose. The screening method was tested on three Brussels sprouts, three cabbage and three cauliflower cultivars, with known reactions to ringspot in the field. Resistance was expressed both in cotyledons and true leaves by a lower number of lesions than the susceptible control and/or by hypersensitive reactions. Results of the seedling tests reflected differences in resistance in the field. Under controlled conditions the new test can be applied year-round to young plants, thus accelerating selection procedures.     

Screening for resistance to canker (Leptosphaeria maculans) in winter oilseed rape (Brassica napus ssp. oleifera)

A test involving infection of seedlings in a glasshouse was evaluated for potential use in screening breeding material by comparing the results with those of field tests. The two tests correlated well for some resistant selections, particularly those closely related to Jet Neuf, and for very susceptible selections. However, two small groups of selections were resistant in the field but consistently susceptible in the glasshouse. Inoculation of mature plants in the glasshouse resulted in similar discrepancies and therefore gave no advantage over the seedling test. These discrepancies limit the usefulness of glasshouse techniques for routine resistance screening. A very high correlation was found overall between internal and external canker symptoms of the crown in nine field trials. Significant differences were found, however, between selections and cultivars in the relative severity of these two symptoms. Upper stem canker and crown canker were found to be very poorly correlated for individual plants, but highly correlated for eultivar means. The significance of these observations is discussed with respect to scoring canker in the field.     

Biotic factors affecting the expression of partial resistance in pea to ascochyta blight in a detached stipule assay

The expression of partial resistance in pea to ascochyta blight (caused by Mycosphaerella pinodes) was studied in a detached stipule assay by quantifying two resistance components (fleck coalescence and lesion expansion) using the method of point inoculation of stipules. Factors determining optimal conditions for the observation of partial resistance are spore concentration, the age of the fungal culture prior to spore harvest and the pathogenicity of the isolate used for testing. Partial resistance was not expressed when spore concentration was high or when the selected isolate was aggressive. Furthermore, assessments of components of partial resistance were highly correlated with disease severity in a seedling test. A screening protocol was developed based on inoculations of detached stipules to study partial resistance in pea. To simplify the rating process, a more comprehensive disease rating scale which took into account fleck coalescence and lesion expansion was tested by screening a large number of genotypes.     

青枯病植物疫苗生产菌FJAT-1458发酵工艺的优化

为提高防治青枯病的植物疫苗菌FJAT-1458的发酵工艺，本文采用液固双相发酵方式，以活菌体数量为指标，通过单因子和正交设计试验对菌株FJAT-1458液体发酵培养基进行优化，并利用农业副产物——微生物发酵床养猪垫料为发酵培养基主成分进行固体培养工艺优化，为研制植物疫苗菌剂提供基础。结果表明，菌株FJAT-1458液体发酵的最适培养基配方为玉米粉2.5%、鱼骨粉0.5%、蔗糖0.1%、蛋白胨0.15%、K₂HPO₄ 0.05%和MgSO₄ 0.025%，发酵48 h后，发酵液活菌含量可达3.87×10⁹ cfu/mL。该菌固体发酵最佳培养基配方为垫料:黄豆饼粉=7:3（质量比），最佳发酵条件为温度35℃、含水量45%、接菌量15%、通气量70%时，培养40 h后，固体菌剂活菌量达4.68×10⁸ cfu/g。     

Leaf position prevails over plant age and leaf age in reflecting resistance to late blight in potato

ABSTRACT The effects of plant age, leaf age, and leaf position on race-nonspecific resistance against Phytophthora infestans were investigated in a series of field and controlled environment experiments with five different potato (Solanum tuberosum) cultivars. Leaf position proved to be the most significant factor; apical leaves were far more resistant to late blight than basal leaves. Plant age and leaf age had only minor effects; therefore, the resistance of a specific leaf remained about the same during its entire lifetime. The gradual increase in late blight resistance from basal leaves to apical leaves appeared to be a general effect, irrespective of cultivar, growing conditions, or resistance test. Therefore, it is important to consider leaf position in tests for late blight resistance, because contrasts in resistance may be ascribed erroneously to differences between genotypes or treatments, whereas they are actually caused by differences in leaf position.     

Evaluation of carrot resistance to alternaria leaf blight in controlled environments

The objective of this study was to find a technique for plant resistance screening to alternaria leaf blight (ALB), caused by the fungus Alternaria dauci , in controlled environments. Glasshouse and laboratory screening methods were compared using three cultivars and F₂ genotypes segregating for ALB resistance evaluated against self-pollinated F₃ field-grown plants. Plant disease was assessed through a disease index obtained from the size and number of symptoms on carrot leaves. The results indicated the value of glasshouse evaluation and the inadequacy of detached leaf and hypocotyl assays for carrot screening for ALB resistance. Spearman's rank correlation, applied to results obtained with both F₂ plants and their progeny, indicated that the optimal evaluation stage for ALB resistance in carrot is 20 days after inoculation. This test was powerful enough to be used as a prescreening test in breeding programmes.     

Development of an effective screening method for partial resistance to Alternaria brassicicola (dark leaf spot) in Brassica rapa

In order to develop a method to measure resistance to Alternaria brassicicola (cause of dark leaf spot disease) in Brassica rapa, the effects of inoculum concentration, leaf stage, leaf age and incubation temperature of inoculation on infection were studied under controlled conditions using several B. rapa genotypes. Three inoculation methods (cotyledon, detached leaf and seedling inoculation) were evaluated for this purpose. The detached leaf inoculation test was the most suitable for screening B. rapa genotypes because clear symptoms were observed on the leaves in less than 24 h, and there was a significant positive correlation between the results from the detached leaf inoculation test and the seedling inoculation test, an established method considered to yield reliable results. In addition, it was very easy to screen plants for resistance on a large scale and to maintain standard physical conditions using detached leaves. For successful infection, inoculum concentration should be adjusted to 5 × 10⁴ conidia ml⁻¹, and incubation temperature should be between 20 °C and 25 °C. The 3rd/4th true leaves from 30 day-old plants were optimal for inoculation. In a screening test using 52 cultivars of B. rapa, the detached leaf test effectively discriminated between various levels of partial resistance among cultivars. As a result, we identified two cultivars, viz Saori and Edononatsu, as highly resistant and five cultivars, viz Tokinashi Taisai, Yajima Kabu, Purara, Norin-F₁-Bekana and Tateiwa Kabu, as having borderline resistance.     

A Reliable Method for Testing the Sensitivity of Botryotinia fuckeliana to Anilinopyrimidines In Vitro

Cyprodinil is a representative of the new class of broad-spectrum anilinopyrimidine fungicides. The effect of cyprodinil on mycelial growth of Botryotinia fuckeliana on solid agar medium depends on the composition of the medium and on the age of the mycelium to be used for the bioassay. An in-vitro method was developed to study the sensitivity distribution to cyprodinil in two wild-type populations of B. fuckeliana from fruits of strawberry with grey mould. To validate the applicability of the method, sensitivities to cyprodinil, mepanipyrim and pyrimethanil were monitored in populations of B. fuckeliana from grapes with grey mould from different vineyards, including one trial vineyard where reduced performance of cyprodinil had been encountered. The monitoring procedure was based on the inhibition of the mycelial growth on a synthetic medium containing L -asparagine (asp-agar) amended with the active ingredients. The mycelium was grown on asp-agar discs, starting from a spore suspension, for 17 h prior to the beginning of the test. This procedure proved to be efficient. The two wild-type populations from different sampling sites showed similar distributions of the sensitivity to cyprodinil. Some isolates from the trial site with reduced performance of anilinopyrimidines showed reduced sensitivities to cyprodinil, mepanipyrim and pyrimethanil, demonstrating cross-resistance between these anilinopyrimidines.     

Screening for resistance to Diaporthe toxica in lupins by estimation of phomopsins and glucoseamine in individual plants

Immunological and biochemical assays were developed for screening for resistance to Diaporthe toxica in individual plants of narrow-leafed lupins ( Lupinus angustifolius ). The former was an enzyme-linked immunosorbent assay (ELISA) for measuring phomopsin mycotoxins and the latter gave an estimation of glucoseamine in infected stem pieces. Stems of L. angustifolius seedlings were inoculated with conidia from D. toxica cultures and, as expected with this latent disease, remained symptomless for 21 days after inoculation. At this time, phomopsins were measured in excised stems that had been incubated for 6 or 8 days to allow mycelial growth from latent infection structures, thereby increasing the phomopsins to detectable levels in individual plants. The estimation of glucoseamine was carried out on the same stems that had been assayed for phomopsins. The method was based on the alkaline deacetylation of chitin to chitosan, the glucoseamine residues of which are de-aminated with nitrous acid, yielding an aldehyde which is determined colorimetrically. At six days after excision, both tests clearly distinguished the very resistant, resistant, intermediate and susceptible lines and they may be useful in large-scale resistance screening in lupin breeding programmes. The ELISA of phomopsins is easier to use and would be particularly useful in the elimination of susceptible plants and those plants expressing intermediate levels of resistance during early generations of the breeding programme.     

麦田猪殃殃对苯磺隆抗药性的快速检测

以采自山东、河南、安徽、陕西等地麦田及非农田的10个猪殃殃Galium aparine生物型为试材,运用单剂量甄别技术对不同生物型对苯磺隆的抗药性进行了检测,并以温室盆栽法及皿内抗性水平测定法验证其可靠性和可行性。结果表明:在甄别剂量(有效成分)85 mg/L下,猪殃殃不同生物型萌发率存在显著性差异,可以较好区分猪殃殃抗、感生物型;温室盆栽法及皿内抗性水平测定法结果一致,除采自山东泰安和陕西周至的麦田猪殃殃对苯磺隆仍处于敏感状态外,其他3个地区麦田猪殃殃均产生了不同程度的抗药性,且均与皿内单剂量甄别法结果一致,表明皿内单剂量甄别法具有可靠性和可行性。     

卵孢白僵菌NEAU30503固态培养条件优化

为了提高卵孢白僵菌NEAU30503在固态培养中的产孢量,采用单因素筛选试验和响应面法对卵孢白僵菌NEAU30503固态发酵条件进行优化。在单因素试验确定最适含水量、接种量、培养温度和培养时间的基础上,应用Box-Behnken试验设计和响应面分析方法优化出最佳固态培养条件为:固态培养基含水量为55%,接种量为15mL/100g,培养温度为27℃,培养时间为7.5d,在此条件下卵孢白僵菌NEAU30503烘干前单位产孢量达到36.72×10~8孢子/g。此方法适用于小型企业和生产单位对白僵菌的快速生产。     

大豆种质资源对大豆孢囊线虫3号和4号生理小种的抗性鉴定

选育和利用抗病品种是控制大豆孢囊线虫病的最经济有效方法.本研究通过测定国内外大豆种质资源对大豆孢囊线虫2个生理小种的抗性水平,以期获得抗大豆孢囊线虫的品种与种质资源.2009年9月-2010年6月,采用PVC柱法在温室内鉴定了300份大豆种质资源分别对大豆孢囊线虫(SCN)3号和4号生理小种的抗性.用3号生理小种测定得到了抗性表现免疫的种质资源12份,表现高抗的28份,表现中抗的39份,分别占测试材料的4.0％、9.3％和13.0％;用4号生理小种测定得到了免疫的种质材料8份,高抗的18份和中抗的51份,分别占测试材料的2.7％、6.0％和17.0％.对2个生理小种的毒力差异比较表明,20份大豆种质资源的抗性表现差异明显,4号生理小种的毒力比3号生理小种强.本研究所鉴定出的抗病品种与资源,不仅对农业生产中利用抗病品种合理布局具有指导意义,而且为大豆抗孢囊线虫病的品种培育提供了重要的抗源材料.     

Assessment of partial resistance of potato to, and pathotype and virulence differences in, potato cyst nematodes

Results are reported for several international collaborative experiments which examined methods of assessing degrees of partial resistance in potato and virulence in potato cyst nematode (PCN, Globodera pallida). It was demonstrated that absolute rates of multiplication can be extremely variable on both susceptible and partially resistant clones, even when the same population and test procedures are employed. It was therefore concluded that, on clones with quantitatively inherited resistance (i.e. partially resistant), absolute rates of multiplication cannot be used to separate pathotypes. Expression of these rates as percentages of those on the susceptible controls reduced the absolute differences between tests, but the values obtained were still too variable for statutory use. However, whatever the environment or nematode population used, it was observed that the resistance of the test clones and virulence of the nematode populations were generally ranked in a similar order. The main exceptions to this were: (1) a Petri-dish test where cv. Vantage was less resistant than in canisters or pots and (2) a pot test where cv. Darwina tended to be more interactive with environmental factors than the other test clones. It was also observed that with some populations of PCN in pots the susceptible cv. Bintje was a less good host than cv. Désirée. On the basis of these results it is suggested that certain partially resistant clones should be used as internal references in statutory, recommended list and breeders' assessment tests against which the resistance of the test clones are compared. For international comparability it is necessary that the different centres conducting such tests use the same reference clones and nematode populations, and similar test methods.     

Evaluation Of Tests to Determine Resistance of Zantedeschia spp. (Araceae) to Soft Rot caused by Erwinia carotovora subsp. carotovora

Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in Zantedeschia spp., particularly in cultivars from the section Aestivae. The disease can be partly controlled by cultural measures, but by combining cultural methods with resistant plant material a promising strategy for control of soft rot can be developed. No tests are available for resistance testing in breeding Zantedeschia spp. Therefore, three tests developed for use in potato breeding were adapted for use on eight cultivars of Zantedeschia spp. Variation was found in all three tests. Resistant control cultivar Zantedeschia aethiopica Crowborough scored most resistant in all three tests. Within the section Aestivae, degrees of susceptibility were identified that were in agreement with each other and with field observations, indicating reliability of two of the methods in which tubers were used. The correlation coefficient of these two tests was high. A new non-destructive test method was developed for use on seedlings which involved immersion of leaf disks in a bacterial suspension. The percentage of decayed leaf area was a measure of resistance and results were in general agreement with the other tests. These methods will be useful for breeding for soft rot resistance and performing genetic analyses.     

Rhizoctonia solani的一种选择性培养基

 通过对多种农药、抗菌素、化学试剂和基础培养基的筛选测定,选出了Rhizoctonia solani的一种选择性培养基。该培养基可以有效地运用于自然土壤中植物残体上R.solani的分离。经比较,该培养基的选择性能要优于国外已报道的同类培养基。     

Screening for blast resistance in rice using AMMI models to understand G x E interaction in Guyana

In rice, totally one hundred and three genotypes were evaluated for resistance against blast disease in three hot spot locations of Guyana. The experiments were conducted in spring and autumn seasons of 2015, 2016 and 2017. The modified upland blast nursery (UBN) method was adopted for screening blast resistance. The Additive Main effects and Multiplicative Interaction (AMMI) analysis was applied to understand the interaction between genotype (G) and environment (E). The results of all seasons at three locations showed the consistent expression of high resistance by genotype FL 127 followed by FG12–08 and FG12–273. The AMMI analysis of variance (ANOVA) revealed that, resistance was dominated by the genotype main effect. The magnitude of genotypes by environment (G x E) sum of square (SS) was 3.64 times smaller than genotype SS, and 6.74 times larger than environment SS. The analysis has indicated that the differences of the genotypes across the environment were not substantial. All of the seven environment vectors appeared to be positively correlated. The screening at Onverwagt Back, autumn 2016 (E7) and Black Bush Polder, spring 2015 (E5) were observed to be the most representative environment. This was clearly revealed by smaller average - environment axis (AEA) angle. Likewise, E4 (Canje, autumn 2015) was observed closer to the “ideal test environment”, thus suggested as an ideal environment for blast resistant screening. Also, genotypes clustered near the origin have showed stable blast disease resistant status and contributed little to both G and G x E. These genotypes proposed as useful ones for breeding disease resistant varieties and hybrids in rice.