Evaluation of biocontrol preparations and plant extracts for the control of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> on potato leaves

The potential of biocontrol products and plant extracts for control of late blight on potato plants, caused by Phytophthora infestans was evaluated in detached leaf assays and on potted plants. Based on an initial screening of 22 preparations and plant extracts, the 10 most active treatments were selected for further investigation. In the detached leaf assays the commercial preparations Elot-Vis, Serenade and Trichodex, and plant extracts of Rheum rhabarbarum and Solidago canadensis showed a significant effect on the level of infestation by P. infestans. However, none of the treatments was as effective as copper. In the case of Serenade, the metabolites produced by its active micro-organism, Bacillus subtilis, were demonstrated to be the effective component of the formulation, and not the micro-organism itself. In order to take curative and protective modes of action into account, the test substances were applied 24 h before, or 90 min after inoculation with P. infestans. Generally, better effects were obtained when the applications were made 24 h before inoculation. For defining the optimum time of application, potted plants were treated 72 or 24 h before, and 1 and 24 h after inoculation with P. infestans. In these tests, Trichodex showed no activity, while Elot-Vis gave best results when applied 1 day before inoculation. Serenade and the extracts of R. rhabarbarum and S. canadensis (all at 5% concentration) however, were effective when applied up to 3 days before and just after inoculation with P. infestans. The results of the experiments on potted plants indicated direct effects on the pathogen for all agents except the extract of S. canadensis, but other mode of actions, e.g. induced resistance, could not be ruled out. None of the treatments had a curative effect.     

Differential metabolic response of barley genotypes,varying in resistance,to trichothecene‐producing and ‐nonproducing (tri5−) isolates of Fusarium graminearum

Resistance of barley to Fusarium graminearum was studied using a pair each of resistant and susceptible black and yellow barley lines. The spikelets were inoculated with a trichothecene‐producing isolate, a trichothecene‐nonproducing isolate (tri5^?), or a mock solution. Spikelets were collected 72 h after inoculation and metabolites were analysed using a LC‐hybrid MS system. Metabolite abundances were used to identify the constitutive (RRC) and induced resistance‐related metabolites (RRI). The pathogen virulence factor, DON, and its plant detoxification product, DON‐3‐O‐glucoside (D3G), were also identified and designated as resistance‐indicator (RI) metabolites. The RRC, RRI and RI metabolites were putatively identified. Jasmonic acid was significantly induced in barley following inoculation with a trichothecene‐producing isolate, but not with a tri5^? isolate. The former isolate reduced the induction of both the number and amount of RR metabolites. The metabolites cinnamic acid, sinapoyl alcohol, coniferin, catechin and naringin were identified only in response to the inoculation with a tri5^? mutant. The abundances of p‐coumaric acid, coniferaldehyde and sinapaldehyde increased more in response to the tri5^? mutant than to the trichothecene‐producing isolate. The total amount of DON synthesized and its conversion to D3G varied greatly between the resistant and susceptible black barley, but not in yellow barley. Interestingly, an increase in the amount of total DON produced was associated with a decrease in the conversion of DON to D3G. The roles of RRC, RRI and RI metabolites in plant defence and their further use as potential biomarkers in screening are discussed.     

Biochemical responses associated with common bean defence against Sclerotinia sclerotiorum

The aim of this study was to investigate changes in defence compounds of common bean cultivars with different levels of resistance to the fungus Sclerotinia sclerotiorum and determine the relation of the compounds to pathogen tolerance. The lines were inoculated with the pathogen and assessed for enzymatic and non-enzymatic parameters related to plant defence: peroxidases (POX), polyphenol oxidases (PPO), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), total soluble phenol and lignin contents. Stem tissue samples were collected from two regions of the plant for biochemical analyses. Stem tissue samples were collected from two regions of the plant for biochemical analyses. In the position one, 5 cm of the stem was collected from the region with necrosis caused by the pathogen, and in the position two, 5 cm of the stem was collected from the end of the position one at the times of 12, 24, 48, 72, 96 and 120 h after inoculation (HAI). Greater lignin and total soluble phenol contents and greater induction of POX and SOD activity in inoculated plants in the region near the inoculation (position one) indicate local activation with later signalling for activation of defence mechanisms in other regions of the plant. The genotype with a greater level of resistance was superior to the susceptible one in regard to lignin production and the activities of POX, APX and SOD defence enzymes. These results suggest that a combination of these defence responses in common bean may contribute to greater plant resistance to the pathogen and that these enzymes have potential use in selection of common bean genotypes.     

Unsaturated fatty acids from zoospores of <Emphasis Type="Italic">Sclerospora graminicola</Emphasis> induce resistance in pearl millet

Downy mildew of pearl millet, caused by Sclerospora graminicola, is a devastating disease, resulting in high economic losses in the semi-arid regions of the world. Recently, induction of host plant resistance using biotic and abiotic inducers are included among disease management practices as an eco-friendly approach. Unsaturated fatty acids are considered as a new generation of plant disease resistance inducers. In the present study, six unsaturated fatty acids, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), linolenic acid, linoleic acid and oleic acid, all originally detected in the zoospores of S. graminicola,were applied to seeds of susceptible cultivars of pearl millet to examine their ability to protect against downy mildew under greenhouse and field conditions. In greenhouse experiments, EPA and AA induced a maximum of 78.6% and 76.5% protection, whereas linoleic acid, DHA and linolenic acid provided up to 66.3%, 61.2% and 24.5% protection, respectively. Oleic acid was not effective in protecting pearl millet (only 5.1% protection). A time interval of four days between treatment of seeds and challenge inoculation was required to obtain optimum protection. Plants raised from treated seeds and challenge inoculated at the tillering and inflorescence stages showed enhanced resistance, resulting in higher grain yield compared to untreated plants of the same cultivar. Chitinase activity was found to be higher in susceptible seedlings of pearl millet after treatment with the fatty acids and pathogen inoculation than in seedlings only inoculated with the pathogen. This indicates that host defence responses are activated and thus that induced resistance is involved in the protection observed. The role of unsaturated fatty acids as activators of resistance against downy mildew in pearl millet is discussed.     

Spectral signatures in the UV range can be combined with secondary plant metabolites by deep learning to characterize barley–powdery mildew interaction

In recent studies, the potential of hyperspectral sensors for the analysis of plant–pathogen interactions was expanded to the ultraviolet range (UV; 200–380 nm) to monitor stress processes in plants. A hyperspectral imaging set-up was established to highlight the influence of early plant–pathogen interactions on secondary plant metabolites. In this study, the plant–pathogen interactions of three different barley lines inoculated with Blumeria graminis f. sp. hordei (Bgh, powdery mildew) were investigated. One susceptible genotype (cv. Ingrid, wild type) and two resistant genotypes (Pallas 01, Mla1- and Mla12-based resistance and Pallas 22, mlo5-based resistance) were used. During the first 5 days after inoculation (dai) the plant reflectance patterns were recorded and plant metabolites relevant in host–pathogen interactions were studied in parallel. Hyperspectral measurements in the UV range revealed that a differentiation between barley genotypes inoculated with Bgh is possible, and distinct reflectance patterns were recorded for each genotype. The extracted and analysed pigments and flavonoids correlated with the spectral data recorded. A classification of noninoculated and inoculated samples with deep learning revealed that a high performance can be achieved with self-attention networks. The subsequent feature importance identified wavelengths as the most important for the classification, and these were linked to pigments and flavonoids. Hyperspectral imaging in the UV range allows the characterization of different resistance reactions, can be linked to changes in secondary plant metabolites, and has the advantage of being a non-invasive method. It therefore enables a greater understanding of plant reactions to biotic stress, as well as resistance reactions.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

The novel elicitor COS‐OGA enhances potato resistance to late blight

Phytophthora infestans is the causal agent of potato late blight. This pathogen is usually controlled by fungicides, but new European regulations have imposed changes in crop protection management that have led to a search for alternative control measures. The induction of plant defence responses by elicitors is a promising new strategy compatible with sustainable agriculture. This study investigated the effect of eliciting a defence response in potato against P. infestans using a formulation of the COS‐OGA elicitor that combines cationic chitosan oligomers (COS) and anionic pectin oligomers (OGA). Trials were conducted under greenhouse conditions to assess the ability of COS‐OGA to control P. infestans. The results showed that three foliar applications with this elicitor significantly increased potato protection against late blight in controlled conditions. The activation of potato defence genes was also evaluated by RT‐qPCR during these trials. Two pathogenesis‐related proteins, basic PR‐1 and acidic PR‐2, were rapidly and significantly up‐regulated by the elicitor treatment. Therefore, these results suggest that the COS‐OGA elicitor increases the protection of potato against P. infestans and that this protection could be explained by an increase in the expression of potato defence genes rather than by biocide activity.     

Implication of peroxynitrite in defence responses of potato to Phytophthora infestans

In this study peroxynitrite (ONOO^?) is proposed as an important player in defence responses during the interaction of potato (Solanum tuberosum) and the oomycete pathogen Phytophthora infestans. The potato–avr P. infestans model system exhibited a transient programme of boosted ONOO^? formation correlated in time with the burst of nitric oxide (NO) and superoxide during the first 6 h post‐inoculation (hpi). The early ONOO^? over‐accumulation was not accompanied by TPx gene expression. In contrast, the compatible interaction revealed a 24 h delay of ONOO^? formation; however, an enhanced level of NO and superoxide correlated with TPx up‐regulation was recorded within the earlier stages of pathogen infection. Peroxynitrite over‐accumulation in the susceptible potato coincided with an enhanced level of protein tyrosine nitration starting from 24 hpi. Surprisingly, the nitroproteome profile of the resistant potato did not show any visible difference after inoculation, apart from one band containing subtilisin‐like protease‐like proteins, which appeared 48 h after pathogen attack. An additional pharmacological approach showed that treatment of the susceptible genotype with ONOO^? followed by inoculation with P. infestans contributed to slowing down of the colonization of host tissues by the pathogen via a faster and stronger up‐regulation of the key defence markers, including the PR‐1 gene. Taken together, the results obtained indicate that a precise control of emitted NO and superoxide in cooperation with thioredoxin‐dependent redox sensors in sites of pathogen ingress could generate a sufficient threshold of ONOO^?, triggering defence responses.     

GC-MS metabolite profiling of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> resistant to metalaxyl

Phytophthora infestans is the most important potato pathogen worldwide. Various alternatives have been used to control the pathogen, including continuous applications of phenylamide fungicides which has caused a rapid development of resistance in populations of P. infestans. Despite the importance of the disease, metabolite profiling of fungicide-resistant P. infestans has not been reported. In vitro resistance of Phytophthora infestans isolates to metalaxyl was characterized and metabolic changes in resistant isolates were evaluated at low (0.5 mg/L) and high (100 mg/L) concentrations of the fungicide. About 70% of the isolates tested showed resistance to metalaxyl and a total of 49 metabolites were differently expressed in resistant isolates growing in the presence of the fungicide. Principal components analysis revealed a distinct metabolite profile of resistant isolates exposed to both low and high levels of metalaxyl. The main metabolites responsible for the clustering in both fungicide concentrations included fatty acids such as hexadecanoic and octadecanoic acids, sugars such as glucose and fructose, aminoacids such as proline and valine, and organic acids such as butanedioic and propanoic acids. Potential resistance-related metabolic pathways are mostly involved in the regulation of the pathogen’s membrane fluidity and included the fatty acid biosynthesis as well as the glycerophospholipid metabolism pathways. This is the first metabolomic-based characterization of fungicide resistance in plant pathogens.     

Root infection of potato by Spongospora subterranea: knowledge review and evidence for decreased plant productivity

Information is reviewed on root infection of potato by the plasmodiophorid Spongospora subterranea f. sp. subterranea. This pathogen has long been recognized as the cause of root galls (hyperplasia) and the economically important disease powdery scab on tubers (modified stolons). The significance for plant productivity of the zoosporangium stages of the pathogen in potato roots has only recently begun to be documented. Two experiments are described that assessed effects of S. subterranea root infection on potato plant root function and productivity. A greenhouse experiment measured root function and plant parameters for eight potato cultivars with markedly different susceptibilities to tuber powdery scab. Water uptake and plant growth were reduced by S. subterranea inoculation in all eight cultivars. The magnitudes of these negative effects, and intensities of root hyperplasia, differed among the cultivars, but were not related to respective susceptibilities to tuber powdery scab. A field trial assessed root function and plant productivity for a cultivar (Iwa) that is very susceptible to Spongospora tuber and root diseases. Soil water content beneath uninoculated plants was consistently less than for inoculated plants, indicating that inoculation reduced water uptake (root function). Inoculation reduced shoot and root dry weights, and reduced weight of tubers per plant by 42%. Spongospora subterranea causes three diseases of potato: root membrane dysfunction, root hyperplasia and tuber powdery scab. The root diseases caused by the pathogen are likely to be important both for powdery scab management and for deleterious effects on potato crop yields.     

Effects of pre‐ and post‐treatment with ethephon on gum formation of peach gummosis caused by Lasiodiplodia theobromae

Peach gummosis, caused by Botryosphaeria spp. fungi, is the process of gum accumulation and exudation in plants. Ethephon (2‐chloroethylphosphonic acid) has profound effects on plants, including enhanced production of secondary metabolites and regulation of plant diseases. This study investigates the effects of application of ethephon before and after inoculation with Lasiodiplodia theobromae on gum formation. Gum formation was promoted by ethephon treatment prior to pathogen inoculation, but inhibited by ethephon applied after the pathogen. The inhibitory effect was counteracted by 1‐methylcyclopropane, which is an ethylene signal inhibitor. 1‐methylcyclopropane also promoted gum formation. Exposure of three isolates of Botryosphaeria to ethephon inhibited mycelial growth. Both treatment methods increased the sugar content at 12 and 24 h post‐inoculation (hpi). However, the sucrose, glucose and fructose contents were significantly higher in shoots with ethephon post‐treatment (application of ethephon after the pathogen inoculation) than those in shoots with ethephon pre‐treatment (application of ethephon prior to pathogen inoculation) at 48 and 72 hpi. The expression of two putative senescence‐related genes, SEN2 and SEN4, were significantly enhanced in pre‐ and post‐treated shoots with ethephon at 24, 48 and 72 hpi. Ethephon application also up‐regulated expression of the pathogenesis‐related protein PR4 while down‐regulating PR1a and PR10. The results show that ethephon has a dual function in regulating gum formation by affecting both the peach shoots and the pathogen.     

Overexpression of StRbohA in Arabidopsis thaliana enhances defence responses against Verticillium dahliae

Plant NADPH oxidases are key regulators of plant–microbe interactions and reactive oxygen species (ROS) are essential to plant defences against pathogens. A significant part in the role played by ROS has been ascribed to plant respiratory burst oxidase homologs (RBOHs). In potato (Solanum tuberosum), where RBOHs were previously shown to be involved in wound-induced oxidative burst, we assessed their expression after inoculation with Verticillium dahliae Kleb. and showed that StRbohA was the only homolog to be differentially induced in potato in response to inoculation. In order to investigate the potential role of this gene in plant protection against wilt diseases, we used Agrobacterium-mediated transformation of Arabidopsis to assess the effects of its overexpression on plant responses to V. dahliae. After inoculation with this pathogen, the transformed Arabidopsis line overexpressing StRbohA showed lower disease severity (percent damaged leaf area and vascular discoloration) as compared to the wild type. It also had higher ROS production and more cell death caused by hydrogen peroxide (H₂O₂), compared to the wild type. Suberization of root cells was also more pronounced in the line overexpressing StRbohA, and supports a possible role for StRBOHA in plant resistance to V. dahliae. Together, these findings indicate that overexpressed StRbohA in Arabidopsis enhances the ROS-mediated defence mechanisms against V. dahliae and can be a potential tool to improve plant resistance to this and other soilborne pathogens that cause wilts in economically important crops.     

十一种杀菌剂防治马铃薯晚疫病的适宜施用时期

为了明确当前市场上具有代表性的11种防治马铃薯晚疫病杀菌剂的适宜施用时期,采用人工接种马铃薯叶片的方法,测定了其中5种药剂在接种晚疫病菌前10 d内不同时间施用的预防效果和其中7种药剂在接种后24 h内不同时间施用的治疗效果。结果显示:在保证良好防治效果的前提下,药剂在病原菌接种前保护性施用的适宜时期比在接种后治疗性施用的适宜时期要长。在测试浓度下,接种前5种供试药剂保持100%防治效果的适宜施用时期为接种前3~10 d;而接种后7种供试药剂保持相同防效的适宜施用时期仅为接种后6~12 h,在生产实际中难以操作。研究结果说明,在田间马铃薯晚疫病的化学防治中,为了取得良好防治效果,每次用药均应在病菌侵入之前采用保护性施用,尽量避免在病菌侵入之后进行治疗性施用。     

The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato

The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses.     

基于链特异性RNA-seq的禾谷镰刀菌全生活史转录组分析

为明确植物病原真菌禾谷镰刀菌Fusarium graminearum全生活史的转录组特征和基因表达模式,采用生物信息学技术对其生活史不同阶段15个时期或组织的链特异性RNA-seq数据进行分析。结果表明,共有8 106个基因在所有时期均有表达,为禾谷镰刀菌生活史核心基因,占总基因数的47.2%;有性生殖和侵染过程中表达的基因数相对较多,其中有性生殖后期表达的基因数最多,达15 221个;无性产孢和侵染小麦穗过程中基因表达水平整体较高,而分生孢子中基因表达水平最低。在燕麦培养基上禾谷镰刀菌气生菌丝的基因表达模式与侵染过程中的基因表达模式较为相似,而与营养生长菌丝的基因表达模式差异较大。该菌次生代谢物合成特征酶基因和分泌蛋白基因的表达模式均分成3类,即分别在侵染、营养生长和有性生殖过程上调表达,暗示其在生活史不同阶段的特异性功能。     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.