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1.
Certain phosphate salts are known inducers of systemic acquired resistance (SAR). In the present study, a local spray application of dipotassium hydrogenphosphate (K2HPO4) was effective in inducing a high level of systemic protection in cucumber plants against anthracnose caused by Colletotrichum lagenarium. Resistance induction by K2HPO4 was associated with localized cell death in cucumber leaves treated with the phosphate salt. The cell death observed, subsequently resulted in the appearance of macroscopically visible, necrotic spots. Appearing lesions resembled those provoked by tobacco necrosis virus (TNV) during a hypersensitive response (HR) that leads to pathogen-induced activation of SAR. Phosphate-mediated cell death was preceeded by a rapid generation of superoxide and hydrogen peroxide. As a further consequence of phosphate application, a local and systemic increase in free and conjugated salicylic acid (SA) levels was detected. The phosphate-induced responses were also identified with a similar time range in cucumber leaves that had been pre-inoculated with TNV. In contrast, none of these responses was triggered by application of the commercial plant activator benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (BTH), which nevertheless was highly effective in inducing SAR in cucumber against anthracnose. In conclusion, the chemical SAR inducer K2HPO4 and the biological inducer TNV share some common early steps in signal transduction leading to SAR in cucumber, which differ from those involved in BTH-mediated SAR.  相似文献   

2.
The effect of an extract of Yucca schidigera on the control and infection process of the apple scab pathogen, Venturia inaequalis, was examined and compared with the chemical resistance inducer, acibenzolar-S-methyl (ASM). In seedling assays, both materials significantly reduced apple scab symptoms and pathogen sporulation on leaves and both showed similar control efficacies as the reference treatment, sulphur. Whereas yucca extract and sulphur gave significant inhibition of conidial germination in vitro, ASM did not inhibit germination. Histopathological studies of the infection process of V. inaequalis in apple leaves showed that the yucca extract primarily acted by inhibiting pre-penetration events and penetration itself. In contrast, the ASM treatment significantly inhibited more stages of the infection process (pre-penetration, penetration and post-penetration events). These observations suggest that the yucca extract acted mainly by a direct fungitoxic effect whereas ASM, as expected, acted as a resistance inducer. However, expression studies of two genes encoding the PR proteins, PR1 and PR8, in apple seedlings indicated that yucca extract may also affect plant defence as expression of both genes was up-regulated following yucca treatment, to a level similar to that observed after treatment with ASM. The fungitoxic effect of sulphur on V. inaequalis was also confirmed in this study.  相似文献   

3.
4.
The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10  µ g mL−1, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10  µ g mL−1, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.  相似文献   

5.
Treatment of tobacco with a mixture containing reactive oxygen species (ROS) and salicylic acid (SA) provided greater protection of tobacco against infection by Pseudomonas syringae pv. tabaci than either treatment alone. Synergism in expression from the promoter of the defense gene PR-1a was also observed. Although the ROS hydrogen peroxide and peracetic acid were poor inducers alone, they enhanced the level of -glucuronidase (GUS) activity expressed from the PR-1a promoter when applied with SA to a transgenic plant bearing a PR-1a::GUS fusion. PR-1a expression was not correlated with increased cell death as determined by Evans blue staining. There was no effect on the timing at which expression was increased by the mixture compared with the separate treatments. The mixture of hydrogen peroxide and SA partially mimicked the effect of a commercial product Oxycom that has field efficacy in improving plant performance. Repetitive applications of Oxycom enhanced expression from the PR-1a promoter and the production of the PR-1 protein. Enhanced activity occurred systemically both from aerial applications to single leaves and from root drenches. Root application strongly promoted veinal expression for the PR-1a promoter compared with confluent production in leaves of sprayed seedlings. Application methods and timing may aid in the success of activators of systemic acquired resistance in field conditions.  相似文献   

6.
7.
The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.  相似文献   

8.
The activities of antioxidant enzymes and photosynthetic responses were investigated in tomato (Lycopersicon esculentum L. var.) pre-treated by plant activators and inoculated by Xanthomonas vesicatoria. Plants were sprayed with acibenzolar-S-methyl, ASM [Bion® 50 WG (0.2 g l−1)] and aqueous extract from dry necrotic tissue flour (VLA) of ‘Lobeira’ (Solanum lycocarpum) bush. Four days later, the plants were challenged with a virulent strain of X. vesicatoria, under greenhouse conditions. Tomato leaves were then assessed to determine the activities of the main antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX). A reduction of bacterial leaf spot severity was observed in plants treated by ASM (49.3%) and VLA (31.4%), without any in vitro inhibitory activity over the pathogen. Controls showed decreases in CO2 assimilation, transpiration, photosynthetic rates, and stomatal conductance. Water use efficiency and carboxylation efficiency were strongly affected in ASM- and VLA-treated, in comparison to controls and healthy plants. The tested substances induced increases in SOD and CAT activities in a delayed enzymatic response typical in compatible plant–pathogen interactions. Measured at daily intervals, activities of APX and POX were significantly (ρ0.05) higher in treated plants than in controls, except for APX in ASM-treated plants where no difference was found when compared to controls. Only POX was clearly induced at the earlier stages after spraying the tomato plants with ASM or VLA. Our results suggest that late increases in antioxidant enzyme activities may play a role in mitigating oxidative damage in restoring the photosynthetic imbalance imposed by the expansion of bacterial lesions.  相似文献   

9.
Mature green mango fruits of commercially important varieties were screened to investigate the levels of constitutive antifungal compounds in peel and to assess anthracnose disease after inoculation with Colletotrichum gloeosporioides. High-pressure liquid chromatography was used to quantify the levels of 5-n-heptadecenylresorcinol and 5-n-pentadecylresorcinol in the peel extracts. The fruit peel of the varieties ‘Kensington Pride’ and ‘Keitt’ were observed to have the highest levels of both 5-n-heptadecenylresorcinol (107.3–123.7 and 49.9–61.4 μg/g FW, respectively) and 5-n-pentadecylresorcinol (6.32–7.99 and 3.30–6.05 μg/g FW, respectively), and the fruits of the two varieties were found to have some resistance to postharvest anthracnose. The varieties ‘Kent’, ‘R2E2’, ‘Nam Doc Mai’, ‘Calypso’ and ‘Honey Gold’ contained much lower concentrations of resorcinols in their peel and three of these varieties were found to be more susceptible to anthracnose. Concentrations of 5-n-heptadecenylresorcinol were significantly lower at the ‘sprung’ and ‘eating ripe’ stages of ripening compared to levels at harvest. Concentrations of 5-n-pentadecylresorcinol did not differ significantly across the three stages of ripening. The levels of these two resorcinols were found to be strongly inter-correlated (P<0.01, r2=0.71), with concentrations of 5-n-heptadecenylresorcinol being an average 18 times higher than those of 5-n-pentadecylresorcinol. At the ‘eating ripe’ stage, significant relationships were observed between the concentrations of each type of alk(en)ylresorcinol and anthracnose lesion areas following postharvest inoculation, P<0.001, r2= 0.69 for 5-n-pentadecylresorcinol, and P<0.001, r2= 0.44 for 5-n-heptadecenylresorcinol.  相似文献   

10.
One spray of 0.1 M aqueous solutions of NPK fertilizers on the upper sides of maize leaves 1, 2, and 3, 2–4 h prior to inoculation, induced systemic resistance (ISR) against northern leaf blight (NLB) caused byExserohilum turcicum andPuccinia sorghi which were developed on leaves 4, 5, 6, and 7. ISR was expressed as a reduction in the number and area of lesions ofE. turcicum and in the number of sporulating or non-sporulating pustules ofP. sorghi on leaves 4, 5, 6, and 7. The reduction in the number of NLB lesions ranged from 51% (KH2PO4) to 69% (K2HPO4) and their size reduction ranged from 73% (KNO3) to 91% (K2HPO4) as compared with water prayed plants. The reduction in the number of pustules ofP. sorghi ranged from 66 to 77%. Fertilizers consisting of various combinations of N, P and K in every case induce similar levels of protection in either host-pathogen system. The induced protection was evident regardless of the leaf position or the rate of NPK accumulation in the upper protected leaves. High fresh weight was detected in the induced plants which expressed the greatest induced protection against NLB and common rast. The possible dual use of NPK fertilizers — to supply nutrients to plants foliarly and at the same time to activate the mechanism(s) for induction of systemic protection toP. sorghi andE. turcicum in maize — is discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 1315-E, 1995 series.  相似文献   

11.
In a survey for effective biocontrol agents of Colletotrichum orbiculare, causal agent of cucumber anthracnose, 43 (MBCu series) and 135 (MBPu series) endophytic actinomycete strains were recovered from surface-sterilized organs of cucumber (Cucumis sativus) and pumpkin (Cucurbita moschata) plants, respectively. The strains were cultured with C. orbiculare on IMA-2 agar medium to determine their in vitro antagonistic ability. Eleven strains that strongly inhibited hyphal growth of the pathogen were selected as potent antagonists. Detached cotyledons of cucumber were soaked in a spore suspension of an antagonistic strain 1 day before challenge-inoculation with the pathogen, and six of these antagonists (MBCu-32, 36, 42, 45, and 56, and MBPu-75) significantly reduced the number and size of the lesions on the cotyledons compared to the untreated control. In the same way, these six strains inhibited lesion development on attached leaves of 3-week-old cucumber seedlings. Strain MBCu-56, the most suppressive of the strains, was selected for further tests. Its suppressiveness increased as concentrations increased; pretreatment of leaves with the strain at 107, 108 and 109 cfu/ml suppressed disease by 72, 79 and 93%, respectively. These results strongly indicated that MBCu-56 has strong potential for controlling cucumber anthracnose. Based on the taxonomic characteristics and 16S rDNA sequence, MBCu-56 was identified as Streptomyces sp. With scanning electron microscopy, the substrate mycelia of the strain were seen to colonize the surface of leaves above the cuticle. Some hyphae also penetrated and grew underneath the cuticle.  相似文献   

12.
The potential of acibenzolar-S-methyl (Benzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester, ASM; Bion 50 WG) and of an extract of Hedera helix, to protect M26 apple rootstocks against fire blight was determined under controlled conditions. Marked differences were observed in the rate and extent of multiplication as well as in pathogen cell viability between control and ASM and H. helix-treated rootstocks. Although the pathogen multiplied abundantly in the plant tissue of water-treated rootstocks and showed severe damage, ASM and the plant extract of H. helix applied prior to inoculation with the causal agent of fire blight, E. amylovora (strain 7/74), suppressed disease development and bacterial multiplication. Physiological observations of ASM and plant extract-treated rootstocks indicated that restriction of pathogen colonization in plant tissue was correlated with a pronounced increase of peroxidase (POX) and chitinase activity. Furthermore, physiological changes caused by these treatments in host cells were characterized by POX labeling methods with SDS-Page electrophoresis. Differences in expression of the POX and protein bands were observed in tissues of plants treated with different inducers. POX activity was determined by the presence of three strong bands in plant extract-treated leaves, two strong bands and one very weak band of about 20.1 and 43 kDa were visible in ASM-treated leaves. Evidence is provided that ASM, as well as extract of H. helix are equally capable of inducing of resistance responses in M26 apple rootstock, which result in an increased resistance to E. amylovora—the fire blight pathogen. These findings demonstrate that both treatments have the ability to induce the activation of defense genes leading to the accumulation of structural and biochemical activities at strategic sites, and these can be associated with induction of resistance against fire-blight.  相似文献   

13.
为明确IDD家族IDD4基因在拟南芥Arabidopsis thaliana抵抗灰葡萄孢菌Botrytis cinerea侵染过程中的作用,通过统计病情指数检测拟南芥野生型(wild type,WT)植株、过表达植株IDD4-OE和缺失突变体idd4植株感染灰葡萄孢菌情况,利用组织染色检测叶片细胞死亡和H2O2的积累情况,采用实时荧光定量PCR(real-time quantitative-PT-PCR,qRT-PCR)技术分析灰葡萄孢菌肌动蛋白基因Bc. ACTIN在3种植株叶片中的表达情况,并施加0.1 mmol/L外源水杨酸(salicylic acid,SA)后测定IDD4-OE植株的病情指数。结果显示,不同株系对灰葡萄孢菌的抗性由高到低依次为idd4>WT>IDD4-OE,IDD4-OE植株中病原菌感染部位的寄主细胞死亡程度比idd4植株严重。染色结果表明,病原菌侵染拟南芥后4 h,接种部位已有H2O2积累。qRT-PCR反应结果显示,Bc. ACTINIDD4-OE中比在idd4植株中的表达水平更高,表明灰葡萄孢菌在IDD4-OE植株中的繁殖速率更快。对IDD4-OE植株外源施加SA后,其病情指数、Bc. ACTIN表达量与WT植株间均无显著差异,说明SA能将感病植株的抗性提高至WT植株的水平,表明IDD4作为负调控因子参与了拟南芥对灰葡萄孢菌的抗性调控,SA在其中发挥着重要作用。  相似文献   

14.
A semi-selective medium for isolation of Xanthomonas axonopodis pv. vignicola from cowpea (Vigna unguiculata) plant and soil samples was developed. Twelve carbon and five nitrogen sources were tested with four strains of X. axonopodispv.vignicola, and 25 antibiotics were screened against saprophytes. -cellobiose (10g) was selected as the optimal carbon source. Among the antibiotics, cefazoline inhibited growth of most of the saprophytes with little effect on strains of the pathogen. ,-methionine enhanced growth of X. axonopodispv.vignicola. Boric acid along with ammonium chloride suppressed growth of Pseudomonas fluorescens. The semi-selective medium designated as cefazoline-cellobiose-methionine (CCM) medium contained K2HPO4 1.34g, KH2PO4 0.4g, MgSO4 0.3g, H3BO3 0.2g, NH4Cl 1.0g, -cellobiose 10g, cycloheximide 0.2g, ,-methionine 1.0g, cefazoline 10mg and agar 14g per l of water (pH 7.2). Colonies of X. axonopodispv.vignicola on CCM medium were whitish, round, raised and 0.2–1.8mm in diameter 96h after incubation. CCM medium generally inhibited growth of Pantoea agglomerans, Bacillus subtilis and saprophytes isolated from cowpea leaves. Colonies of Pseudomonas fluorescens and a saprophytic bacterium, which were not completely suppressed by CCM, could be differentiated from X. axonopodispv.vignicola by their smaller size and different color. The CCM medium proved useful for isolation of X. axonopodispv.vignicola from cowpea plant and soil samples. This is the first report of a semi-selective medium developed for detection of X. axonopodispv.vignicola.  相似文献   

15.
Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar-S-methyl), and the rhizobacteriaPseudomonas aeruginosa KMPCH andP. fluorescens WCS417 were tested for their ability to induce resistance toColletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10−3 to 10−7 M BTH 3 days before the challenge inoculation. At a high concentration (10−3 M), BTH induced resistance of the same order as resistance induced by the pathogenC. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg−1 BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants.P. aeruginosa KMPCH induced resistance in bean againstC. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean—C. lindemuthianum system.P.fluorescens WCS417 could induce resistance toC. lindemuthianum in a susceptible and in moderately resistant interactions. http://www.phytoparasitica.org posting Jan. 16, 2002.  相似文献   

16.
A method was developed using scanned computer images and software programs to measure the lesion area on leaves with cucumber anthracnose caused by Colletotrichum orbiculare. After cucumber plants were inoculated with various concentrations of conidia, lesions on diseased leaves were scanned, manipulated with Adobe Photoshop 6.0, and measured using the blob analysis feature in Matrox Inspector 2.2. The method requires relatively low-cost equipment including a scanner, a personal computer, and two programs: Adobe Photoshop 6.0 and Matrox Inspector 2.2. Because stored images are used, the lesion area can be measured as time permits. Processing the images requires about 3 min per sample. The image assessment accurately detected anthracnose lesions on cucumber leaves and could be applied to other foliar necrotic or spot diseases.  相似文献   

17.
In an effort to expand the spectrum of larvicides effective againstLiriomyza huidobrensis (Blanchard), the effect of spinosad was studied on the mortality of the leafminer under laboratory conditions. Bean plants infested with various leafminer stages (egg through third instar) were treated by dipping leaves in a liter of water containing 24, 48 or 96 mg a.i. spinosad or by drenching the soil of plants with 200 ml of water containing 12 mg, 24 mg or 48 mg a.i. spinosad. In general, leaf dipping was more efficacious and adverse effects were observed sooner than with soil drench. All concentrations of spinosad significantly reduced the number of adults that emerged except leaf dip of third instar; only the highest concentration caused significant reduction of pupae and adults. Spinosad would likely be a valuable insecticide for control of the pea leafminer,L. huidobrensis. http://www.phytoparasitica.org posting Nov. 4, 2005.  相似文献   

18.
Secondary metabolites and host defense compounds were shown to occur in xylem sap, and leaves of Vitis vinifera cv. Italia and cv. Matilde naturally infected by the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme). Samples of xylem sap and leaves were collected from healthy vines and from vines showing severe symptoms of brown wood-streaking caused by Pch and Tmi, or from vines with symptoms of both brown wood-streaking and white rot caused by Fme. Xylem sap collection was carried out during the early spring of 2003 and 2004, corresponding to the phenological phases: (A) cotton bud; (B) green tip; (C) leaves out; (D) stretched out leaves; and (E) visible clusters. In the present work we have studied the accumulation of biomolecules (pentaketides and α-glucans), host defense compounds (benzaldehydes, benzoic acid and cinnamic acid derivatives, flavonols, flavanols, flavan-3-ol derivatives and stilbenes) at different stages of grapevine development. Accumulation and changes in total phenolics and recurring phenolics, and of three phytotoxic secondary metabolites (scytalone, isosclerone and pullulan) were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that phenolic concentrations are strongly related to the cv.  相似文献   

19.
Foliar sprays and soil drenches with DL--amino-n-butyric acid (BABA) reduced the number of Heterodera avenae and H. latipons cysts on wheat and barley. Foliar sprays of wheat with 8000mgl–1 BABA reduced the number of H. avenae cysts by 90%, whereas 2000mgl–1 BABA was enough to reduce the number of H. latipons cysts by 79%. Multiple spray treatments with 2000mgl–1 BABA at 10-day intervals reduced the number of H. avenae cysts on wheat and barley. A soil drench of wheat with 125mgl–1 BABA reduced the number of H. latipons cysts by 93% and H. avenae cysts by 43%. Second-stage juveniles of these nematodes penetrated and formed syncytia in wheat roots soil-drenched with BABA. More adult males of H. avenae were produced in BABA (<250mg1–1)-treated wheat roots (~76%) than in untreated roots (27%). Soil drenches with higher concentrations of BABA inhibited development of adult males and females. Several chemical elicitors of induced resistance were tested for their ability to reduce the number of H. avenae cysts on wheat. Only BABA was found to be an effective resistance inducer. The number of egg masses of an unidentified Meloidogyne sp. root-knot nematode, which infects only monocots, was also reduced by 95% by a soil drench of wheat with 500mgl–1 BABA. Development of this nematode inside the BABA-treated roots was also inhibited.  相似文献   

20.
This study aimed to evaluate the effect of silicon (Si) rates on some components of sorghum resistance to anthracnose. Two 2×5 factorial experiments, consisting of two sorghum lines (BR005 and BR009, resistant and susceptible, respectively) and five Si application rates (0, 0.06, 0.12, 0.24 and 0.30 g Si kg−1 of soil) were arranged in a completely randomised design with three replications. Plants from both lines were inoculated with a conidial suspension of Colletotrichum sublineolum (1×106 conidia ml−1) 30 days after emergence. The incubation period (IP), latent period (LP60), area under relative infection efficiency progress curve (AURIEPC), area under anthracnose index progress curve (AUAIPC), final disease severity (FDS), percentage of pigmented leaf area (PLA), and percentage of necrotic leaf area (NLA) were evaluated. Silicon and calcium (Ca) content in leaf tissue of both lines was also determined. The content of Si in leaf tissue increased relative to the control by 55 and 58%, respectively, for the susceptible and resistant lines. There was no significant change in Ca content in leaf tissue for either of the lines; therefore the variations in Si accounted for differences in the level of disease response. The IP for the resistant line was not affected by Si application rates. The LP60 was not evaluated in the resistant line due to the absence of acervuli. For the resistant line, Si application rates had no significant effect on AUAIPC, FDS, percentage of PLA, and percentage of NLA. On the susceptible line, a quadratic regression model best described the effect of Si application rates on IP, LP60, AURIEPC, AUAIPC, FDS, percentage of PLA, and percentage of NLA. The correlation between Si content in leaf tissue of the susceptible line and the AURIEPC, AUAIPC, FDS, PLA, and NLA was negatively significant (r = −0.57, −0.37, −0.40, −0.67, and −0.77, respectively). There was no correlation between Si content and IP or LP60. The correlation between the percentage of PLA with the percentage of NLA was negatively significant (r = −0.74). In conclusion, the results from this study underscore the importance of Si in sorghum resistance to anthracnose particularly for the susceptible line.  相似文献   

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