Effectiveness of systemic resistance in bean against foliar and soilborne pathogens as induced by biological and chemical means

Unifoliate leaves of 9-day-old green bean, Phaseolus vulgaris cv. Redlands Pioneer, were inoculated with 10⁴ conidia/ml Colletotrichum lindemuthianum , causing local lesions, or sprayed with 20 μg 2, 6-dichloro-isonicotinic acid/ml formulated by Ciba-Geigy Ltd as CGA 41396. At various times afterwards (7–16 days), first, second or third trifoliate leaves of these plants were challenge-inoculated with 10⁵ conidia/ml C. lindemuthianum or with the rust pathogen, Uromyces appendiculatus. The numbers of anthracnose lesions or rust uredinia resulting from challenge-inoculation were reduced to similar extents by both pre-treatments compared with control plants. Halo blight, caused by Pseudomonas syringae pv. phaseolicola , was reduced in first trifoliates following treatment of unifoliate leaves 6 days earlier with CGA 41396. Induced resistance to root-infecting pathogens was not observed when stems of either 14- or 16-day-old plants were inoculated with mycelial plugs of Fusarium solani f. sp. phaseoli , or when 11- and 15-day-old plants were inoculated with Rhizoctonia sp., Treatment with CGA 41396 did not protect seedlings when they were transplanted into a mix containing the Fusarium sp. 1 day later.     

Chlorophyll fluorescence imaging: a new method for rapid detection of herbicide resistance in Alopecurus myosuroides

Due to the steadily increasing number of putative herbicide‐resistant weed populations, the demand for rapid in‐season tests is rising. In this study, we introduce a new quantitative herbicide‐resistance test system based on chlorophyll fluorescence imaging analysis of photosynthesis‐related parameters. Susceptible and herbicide‐resistant populations of Alopecurus myosuroides (black‐grass) were cultivated in multiwell tissue culture plates containing nutrient agar and different dosages of fenoxaprop‐P‐ethyl and mesosulfuron+iodosulfuron. The maximum quantum efficiency of the PSII was measured 3 h after transplanting (HAT) and then for seven days every 24 h. Data of maximum quantum efficiency of the PSII were compared with standard whole‐plant pot tests and molecular tests for target‐site mutations. It was possible to fit dose‐response curves and calculate corresponding resistance factors for ED90 for all populations tested using the chlorophyll fluorescence imaging. It was possible to distinguish between resistant and susceptible populations. The results of the chlorophyll fluorescence imaging corresponded well with the standard whole‐plant pot tests in the glasshouse. However, populations with proved target‐site mutations did not differ from other herbicide‐resistant populations in the maximum quantum efficiency values of the PSII. We conclude that the chlorophyll fluorescence imaging provides reliable data on herbicide resistance for both modes of action tested in a shorter time and using less space, compared with standard whole‐plant pot tests in the glasshouse.     

Testing the systemic induced resistance hypothesis with Austrian pine and Diplodia sapinea

Systemic induction of defenses (e.g. phenolic metabolites) is considered vital in conifer resistance to pathogens and insects, and forms the mechanistic basis of the systemic induced resistance hypothesis (SIRH). In this study, the SIRH was tested on juvenile Austrian pine. Main stems expressed SIR in a manner that was consistent with the SIRH, while shoots became uniformly more susceptible to subsequent inoculations, demonstrating clear organ specificity in the tree's response. The majority of phenolic metabolites were poorly correlated with phenotype. Thus, the defensive system of Austrian pine is highly plastic and organ specific, and cannot be predicted by phenolic profiles alone.     

Menadione sodium bisulphite: a novel plant defence activator which enhances local and systemic resistance to infection by Leptosphaeria maculans in oilseed rape

Pretreatment of the first true leaves of oilseed rape plants ( Brassica napus cv. Bristol) with menadione sodium bisulphite (MSB) locally and systemically induced resistance, as shown by reduced lesion size and number, to infection by the fungal pathogen Leptosphaeria maculans , the causal agent of stem canker. Using a known systemic activator of salicylic acid-dependent PR-1 induction, acibenzolar- S -methyl (BTH; S -methylbenzo[1,2,3]thiadiazole-7-carbothiate) as a comparison, real-time PCR expression analysis of genes encoding a pathogenesis-related protein 1 ( PR-1 ) and an ascorbate peroxidase ( APX ) demonstrated a systemic enhancement of APX expression in MSB-pretreated plants, with no effect on PR-1 expression, suggesting augmented reactive oxygen species production in MSB-pretreated plants. The results demonstrate MSB to be an effective resistance activator in oilseed rape, and potentially useful for the control of stem canker.     

Protection of Brassica seedlings against downy mildew and damping-off by seed treatment with CGA 245704, an activator of systemic acquired resistance

Control of seedling diseases is a major priority in many crop systems. Seed treatments that induce systemic resistance after seedling emergence may be an ideal way to provide protection against disease during the establishment of the crop. CGA 245704, a chemical activator of systemic acquired resistance, was tested as a seed treatment against two Brassica diseases with contrasting infection biologies, the airborne downy mildew pathogen, Peronospora parasitica, and the soilborne fungus, Rhizoctonia solani. Seeds of two Brassica spp. were either imbibed with various concentrations of the compound or imbibed and then dried. Both the imbibition treatment alone and the imbibition treatment followed by seed drying had a significant effect on the sporulation intensity of P. parasitica for all concentrations of the compound used, whereas the imbibition treatment provided some control of damping-off caused by R. solani, with the degree of control being highly dependent on the concentration applied to the seed. Seed treatment with the plant activator CGA 245704 might therefore simultaneously control several seedling diseases, thereby providing a novel option for management of these diseases. © 1998 SCI.     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

The role of fungal endophyte infection in the evolution of Lolium multiflorum resistance to diclofop-methyl

Diclofop-methyl resistance was evaluated in populations of Italian ryegrass ( Lolium multiflorum Lam.) infected and uninfected with fungal endophyte ( Neotyphodium ). Survival was tested in susceptible populations using herbicide screening. The results served as inputs to a model for investigating the role of endophyte infection in the evolution of L. multiflorum resistance to herbicide. The tolerance of infected plants varied depending on the origin of the population and the herbicide dose. Only in some populations and at some diclofop-methyl doses did plants infected with these endophytes have higher survivorship than endophyte-free plants. The model demonstrated that endophytes might indeed play an important role in the evolution of herbicide-resistant weeds, delaying the appearance of herbicide resistance.     

Beta-cyanoalanine synthase as a molecular marker for induced resistance by fungal glycoprotein elicitor and commercial plant activators

ABSTRACT The biocontrol agent Pythium oligandrum produces glycoprotein elicitor in the cell wall fraction, designated CWP, and induces resistance to a broad range of pathogens. To understand the mechanism of CWP-induced resistance to pathogens, gene expression at the early stage of CWP treatment in tomato roots was analyzed using a cDNA array. At 4 h after CWP treatment, 144 genes were up-regulated and 99 genes were down-regulated. In the 144 up-regulated genes, nine genes exhibited about eightfold increased expression. Analysis of the response of these nine genes to three commercial plant activators indicated that a high level of one gene, beta-cyanoalanine synthase gene (LeCAS) encoding hydrogen cyanide (HCN) detoxification enzyme, was stably induced in tomato roots by such treatment. However, expression of LeCAS was not significantly induced in tomato roots at 4 h by abiotic stresses, whereas only a very low level of induction of such expression by cold stress was observed. This LeCAS expression was also induced after exogenous treatment with a low level of 1-amino-cyclopropane-1-carboxylate as the precursor of ethylene, but not with either salicylic acid or methyl jas-monate. The induction of LeCAS expression in CWP-treated and plant activator-treated roots is likely to be caused by the detoxification of HCN during ethylene production. Transient activation of LeCAS expression caused by ethylene production in tomato roots may be a general phenomenon in fungal elicitor-induced and synthetic plant activator-induced resistance. LeCAS seems to be useful for screening possible novel plant activators for plant protection against pathogens.     

Activation of systemic disease resistance in pea by an avirulent bacterium or a benzothiadiazole, but not by a fungal leaf spot pathogen

Inoculation of first expanded leaves of pea seedlings with an avirulent strain of Pseudomonas syringae pv. pisi , or treatment with sprays of a benzothiadiazole (20 or 100  μ g a.i. mL⁻¹), decreased the susceptibility of subsequent leaves 7 or 14 days later to challenge inoculation with Mycosphaerella pinodes . Inoculation of first leaves with a virulent strain of P. syringae pv. pisi or with M. pinodes did not decrease the susceptibility of plants to M. pinodes . Treatments effective in decreasing susceptibility to M. pinodes were similarly active against Uromyces viciae-fabae and virulent P. syringae pv. pisi . Effective treatments also enhanced the activities of the enzymes β-1,3-glucanase and chitinase in untreated upper leaves 6 days later. Ineffective treatments for decreased susceptibility had no effect on the activity of the enzymes. None of the treatments enhanced peroxidase activities. The results are discussed in relation to the reported signalling effects of the benzothiadiazole and in relation to a suggested high activity of the avirulent P. syringae pv. pisi strain and inactivity of M. pinodes in enhancing natural signalling.     

Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment

Melon plants locally infected with Colletotrichum lagenarium display a marked increase in chitinase activities (exo- and endo-activities) throughout the whole plant. This increase begins 3 days after inoculation in the inoculated cotyledon, and then occurs sequentially in the non-infected tissues.Both fungal elicitors and plant endogenous elicitors induce a rapid increase in chitinase activity in the treated cotyledon. In other organs, chitinase activity is stimulated, to a lesser extent and after a lag period, only by fungal elicitors.The earlier, more rapid, systemic induction of chitinase activity, produced by treatment with the fungal elicitor is correlated by the increased resistance of the tissues to infection by the pathogen.     

Increased activities of ribonuclease and protease after challenge in tobacco plants with induced systemic resistance

Inoculation of 3-4 lower leaves of tobacco with tobacco mosaic virus (TMV) increased ribonuclease (RNase) and protease activities in inoculated leaves. Little or no increase in the activities were apparent in upper noninoculated leaves prior to challenge. After challenge with TMV or Peronospora tabacina, RNase activities increased more rapidly in the upper leaves of induced plants as compared to those of noninduced plants. Protease activities in the leaves challenged with P. tabacina or TMV also increased after challenge, but little or no differences in the activities were apparent between induced and noninduced plants. The incubation of purified TMV with leaf extracts obtained from induced challenged, noninduced challenged and noninduced unchallenged plants prior to inoculation did not affect the number of local lesions formed on tobacco plants.     

A fungal elicitor enhances the resistance of tomato fruit to Fusarium oxysporum infection by activating the phenylpropanoid metabolic pathway

Fungi infection in fruits is an important factor in postharvest losses. The effect of a treatment with a fungal elicitor on the response of tomato fruit to Fusarium oxysporum infection and changes in the phenylpropanoid metabolic pathway was studied. Fungal elicitor retarded for 3 days the development of the Fusarium rot development in tomato, at concentration of 2 g?l ^?1 (B2-F treatment). This treatment also induced a 3.11- and 6.03-fold increase of caffeic and chlorogenic acids, respectively, as compared with the control. Furthermore, the flavonoids naringenin-7-O-glucoside, rutin and kaempferol-3-O-glucoside, showed a greater abundance in tomato under the B2-F treatment after 6 days at 20°C. It is concluded that the fungal elicitor reduced the development of Fusarium rot by inducing the biosynthesis of metabolites from the phenylpropanoid metabolic pathway which forms part of the defense response in tomato fruit.     

Chlorophyll fluorescence for visualizing the spatial and temporal spread of Phytophthora alni subsp. alni in alder bark tissue

The impact of alder Phytophthora (Phytophthora alni subsp. alni) on corticular photosynthetic metabolism was explored via measurements of chlorophyll fluorescence. Stem inoculation induced a sharp reduction of maximum (F_v/F_m) and effective quantum yield of PSII (). Observations of the axial and radial spread of the pathogen revealed that near to the point of inoculation and in the whole centre of the stem lesion F_v/F_m and of the cortex chlorenchyma decreased to almost zero, indicating tissue necrosis. Low values of F_v/F_m and were also found in some presymptomatic regions beyond the visible stem lesion. In contrast, substantial photosynthetic activity was found in uninvaded parts of the inoculated trees and in the control. These stem parts showed a marked light‐adapted quantum efficiency of PSII as well as marked electron transport rates in their bark tissues. Thus, corticular photosynthesis stayed unaffected in these stem parts supporting stem carbon balance. Chlorophyll fluorescence measurements in the field illustrated that stem infection with P. alni subsp. alni and the effect on the bark tissues is not only highly heterogeneous but also underlies very quick temporal changes, due to a rapid destruction of the photosynthetic apparatus. The results show that chlorophyll fluorescence measurements and fluorescence imaging are useful indicators of tissue infection caused by Phytophthora infection of bark tissues. Chlorophyll fluorescence measurements can be used to map and visualize the spatial as well as temporal spread of bark pathogens and can give a first indication of invasion of the host tissue beyond the visible lesion.     

A green fluorescent protein-based screening method for identification of resistance in anthurium to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae

Resistance of cultivars of Anthurium andraeanum to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae , the causal organism of bacterial blight disease of anthurium, was investigated using a bioengineered bacterial strain containing p519ngfp plasmid. Successful infection establishment in anthurium was found to be cultivar and inoculum density dependent, but independent of plant age. Injection of cut petioles (stage-2 leaf) with 100  µ L inoculum (10⁹ CFU mL⁻¹) resulted in 100% infection establishment in susceptible cultivars on a repeatable basis, and differentiated between various levels of observed field resistance. Time to death (weeks) and proportion of dead plants best differentiated between levels of resistance and cultivars were placed in four groups based on these criteria. The susceptible group (32 cultivars) rapidly declined within 6–12 weeks of inoculation (WAI) and resulted in 100% plant death; the moderately resistant group (10 cultivars) declined within 12 WAI, but resulted in less than 100% plant death; the resistant category had less than 100% plant death with a slow decline taking over 20 weeks; and the highly resistant category (15 cultivars) showed 0% infection. The correlation coefficient between green fluorescent protein (GFP)-fluorescence and eventual death of plants was 0·90, indicating that the final death of individual plants can be reasonably well predicted based on GFP-fluorescence data at 5 WAI. Hence GFP data at 5 WAI can be used for early detection of latently infected plants and may assist screening for resistance in segregating populations of anthurium.     

Induction and expression of systemic resistance to the anthracnose disease in bean

Development of anthracnose symptoms in trifoliate leaves of Phaseolus vulgaris inoculated with Colletotrichum lindemuthianum was greatly restricted when the primary leaves had been inoculated with the same pathogen 1 week earlier. Penetration from appressoria was inhibited and hyphal growth in epidermal cells was sometimes decreased in preinoculated plants. The response of preinoculated plants also differed: wall appositions and encroachment of nuclei at attempted penetration sites occurred more frequently, and earlier cell death often followed successful penetration. Resistance in trifoliate leaves was also induced by injection of primary leaves with conidia or culture filtrates of the pathogen or a dialysis retentate of these filtrates. Resistance in epicotyls and primary leaves was induced by inoculation of the hypocotyl 1 week earlier. Inoculated cotyledons abscissed, and failed to alter the susceptibility of primary or trifoliate leaves to subsequent inoculation. The results are discussed in relation to a hypothesis to explain the process of induced systemic resistance.     

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.     

我国农业有害生物抗性风险评估与治理进展

20世纪80年代中期以来,全国不少科研、教学、推广单位,对农作物害虫(螨)、病原菌和杂草对农药及转基因作物抗性的风险评估与综合治理,进行了大量的协作研发和推广工作。本文综述其研发、推广成果,并展望了未来的发展方向。     

TaqMan探针实时荧光PCR快速检测苹果牛眼果腐病菌方法的建立

苹果牛眼果腐病菌(Neofabraea malicorticis、N.perennans、N.alba和N.kienholzii)是我国检疫性植物病原真菌,为建立该病菌的实时荧光PCR检测法,根据病菌及其近缘种的翻译延伸因子(EF-1α)的保守序列设计了特异性探针,分别以苹果牛眼果腐病菌菌株和本研究构建的EF-1α重组质粒DNA为阳性标准品检验探针的特异性和灵敏度。结果显示探针MAL-P、PER-P、ALB-P和KIE-P分别对N.malicorticis、N.perennans、N.alba和N.kienholzii表现特异性阳性扩增,而与近缘种及其他常见的果腐病菌无交叉反应,单重探针和4种探针混合液的灵敏度分别达1 fg/μL和10 fg/μL DNA。总计从美国、智利、新西兰、法国进境截获的29批可疑病果的分离物中检测到PCR阳性荧光信号,包括20批N.perennans、8批N.alba和1批N.kienholzii。该方法在6 h内即可完成整个检测流程,其特异性强、灵敏度高,适用于实际样品中苹果牛眼果腐病菌的快速检测。