Fusarium oxysporum Fo47 confers protection to pepper plants against Verticillium dahliae and Phytophthora capsici,and induces the expression of defence genes

The application of the nonpathogenic isolate Fusarium oxysporum 47 (Fo47) reduced the symptoms of verticillium wilt, phytophthora root rot and phytophthora blight in pepper plants. Botrytis cinerea was also tested on the leaves of plants treated with Fo47, but no protection was observed. Verticillium dahliae colonies cultured in the presence of Fo47 grew slower than control cultures, but Phytophthora capsici growth was unaffected by Fo47. At least part of the protection effect observed against V. dahliae could therefore be due to antagonism or competition. In order to search for induced resistance mechanisms, three defence genes previously related to pepper resistance were monitored over time. These genes encode a basic PR‐1 protein (CABPR1), a class II chitinase (CACHI2) and a sesquiterpene cyclase (CASC1) involved in the synthesis of capsidiol, a phytoalexin. These three genes were transiently up‐regulated in the roots by Fo47 in the absence of inoculation with the pathogen, but in the stem only CABPR1 was up‐regulated. In plants that were inoculated with V. dahliae after the Fo47 treatment, the three genes had a higher relative expression level than the control in both the roots and the stem.     

Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants

Two bacterial isolates and one strain of Trichoderma harzianum were tested alone and in combination with chitin for efficacy in control of root rot disease caused by Phytophthora capsici and Rhizoctonia solani in pepper plants under greenhouse conditions. These bacteria (Bacillus subtilis HS93 and B. licheniformis LS674) were isolated from repeatedly washed roots of pepper plants. In in vitro assays, HS93, LS674 and T. harzianum were antagonistic against P. capsici and R. solani and produced high levels of chitinase. Seed treatment and root drenching with bacterial suspensions of HS93 with 0.5% chitin was more effective against Phytophthora and Rhizoctonia root rot than addition of the organisms without chitin. LS674 and T. harzianum reduced Rhizoctonia but not Phytophthora root rot. In two greenhouse tests, seed treatment and root drenching with HS93 amended with chitin enhanced its biocontrol activity against P. capsici but not on R. solani. The effects of LS674 and T. harzianum against R. solani were significantly enhanced when they were used as suspensions with 0.5% chitin for root drenching, but this had no effect on P. capsici. In both greenhouse experiments, the use of 0.5% chitin alone for root drenching reduced Rhizoctonia root rot. Reduction of root rot disease was accompanied by increased yield. These results show that the antagonistic activity of HS93, LS674 and T. harzianum may be stimulated by chitin resulting in significant improvements in their effectiveness against pathogens.     

Induced resistance againstPhytophthora capsici in pepper plants in response to DL-β-amino-n-butyric acid

Treatment of pepper plants with the nonprotein amino acid, DL-ß-amino-n-butyric acid (BABA) induced resistance to subsequent infection byPhytophthora capsici. In contrast, theα-, andγ-isomers of aminobutyric acid were ineffective as inducers of resistance. A relatively high concentration of BABA at 1,000μg ml^?1, which had no antifungal activityin vitro againstP. capsici, was required to induce resistance against Phytophthora blight with a foliar and stem spray, thus leading to complete control of the disease. About 1 day interval between BABA-treatment and challenge inoculation was sufficient to induce resistance in pepper plants. High inoculum levels ofP. capsici caused Phytophthora development slowly in pepper stems treated with BABA, especially at early plant growth stage, which suggests that the induced resistance in pepper plants may be more quantitative rather than qualitative. BABA applied to the root system also protected pepper stems fromP. capsici infection.     

An ultrastructural study of the effect of metalaxyl on Phytophthora capsici infected stems of Capsicum annuum

Stems of pepper seedlings were inoculated with zoospores of Phytophthora capsici 18 h after a soil-drench with metalaxyl solution (5 μg ml^?1) or water. Infected stem tissues were examined by electron microscopy 24 h after inoculation. Compared with untreated controls, in which the fungal cells were generally normal in shape and ultrastructure, the most conspicuous effects of metalaxyl treatment on the fungal ultrastructure in the stem tissue were an abnormal shrinking of the fungal cell, a separation of the plasma membrane from the hyphal wall, a peculiar invagination and breakdown of the plasma membrane, the presence of vesicles in the invaginated spaces and within the damaged fungal cells, and an indistinct structure of cell organelles. In metalaxyl-treated stems, an electron-dense material was apposed in those sites of the host cell wall in most intimate contact with the fungal cell wall, indicating that the deposition of these substances from the host cell walls may function as a plant defence reaction to the fungus, whereas in untreated controls, the dark-stained host cytoplasm did not aggregate around the sites of fungal contact. The haustorium was encased by the extra-haustorial matrix in treated stems. These results suggest that metalaxyl treatment not only changes the fine structure of P. capsici, but may also induce the plant defence reaction.     

Integrated pest management: A strategy to control resistance of Spodoptera exigua and Helicoverpa armigera caterpillars to insecticides on soybean in the Mekong Delta

The antibiotic nucleoside tubercidin produced by Streptomyces viola-ceoniger was evaluated for in-vivo efficacy and in-vitro activity against Phytophthora capsici, Magnaporthe grisea and Colletotrichum gloeosporioides. Tubercidin was more effective against P. capsici and M. grisea than against C. gloeosporioides in inhibiting mycelial growth. The bioassay on TLC plates was the most sensitive method and allowed the evaluation of antifungal activity of tubercidin even at a low concentration of 0.1 μgml^?1. As compared to the systemic fungicide metalaxyl, tubercidin was similar or somewhat higher in inhibition of mycelial growth of P. capsici. When applied to pepper stems, tubercidin was equally as effective as metalaxyl in the control of phytophthora blight in pepper plants, irrespective of application time and concentration. The treatment with 1000 μg ml^?1 tubercidin induced phytotoxicity in pepper plants. No control efficacy of phytophtora blight was observed in pepper plants supplied with a soil drench of tubercidin. Treatment with tubercidin at 500 μg ml^?1 completely protected pepper plants at first branch stage from phytophthora blight until four days after application. The control efficacy of tubercidin drastically declined seven days after application.     

Phenylalanine ammonia lyase activity in chilli CM-334 infected by Phytophthora capsici and Nacobbus aberrans

We tested the hypothesis that PAL activity in chilli plants CM-334 inoculated with Nacobbus aberrans (Na) alone or in combination with Phytophthora capsici (Pc), is lower than in those inoculated only with Pc. At 21 days after nematode inoculation, inoculated plants showed a significant (P < 0.01) reduction of 48% in PAL activity compared to those non-inoculated in two separate experiments. In two other tests, where plants were inoculated with the oomycete 21 days after inoculation with the nematode, PAL activity at 2, 4, 6, 8 and 24 h after inoculation with Pc was significantly higher (Tukey, P < 0.01) in plants inoculated only with Pc than in plants inoculated only with Na or both pathogens (Na+Pc).     

Mycorrhizal protection of chili plants challenged by <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Hydrogen peroxide (H₂O₂) has been implicated in many stress conditions. Control of H₂O₂ levels is complex and dissection of mechanisms generating and relieving H₂O₂ stress is difficult, particularly in intact plants. Here the role of the mycorrhizal inoculation in chili plants challenged with Phytophthora capsici was investigated to study the effect on hypersensitive response. In the treatment without mycorrhiza (treatment T3) and with mycorrhiza (considered treatment T4) visible disorders were detected two days after inoculation with P. capsici, but in the next days T3 plants rapidly developed 25% more necrotic lesions on the leaves than T4 plants. Leaf necrosis correlated with H₂O₂ accumulation and the greater damage observed in T3 plants coincided with larger accumulation of H₂O₂ after 12 h of inoculation accompanied with an increase in POX (peroxidase) and SOD (superoxide dismutase) activity. T4-infected and mycorrhizal plants exhibited an earlier accumulation of H₂O₂ starting 6 h after inoculation with lower levels compared to T3 plants. Correlated with observed damage, POX and SOD activity measured in T4 plants indirectly suggest a smaller accumulation of ROS (reactive oxygen species) leading to a decrease in the wounds observed and slightly diminishing the advance of the pathogen. According to these findings, we conclude that mycorrhizal colonization contributes significantly in maintaining the redox balance during oxidative stress, but the exact mechanism is still uncertain.     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Plant growth enhancement and disease control byTrichoderma harzianum in vegetable seedlings grown under commercial conditions

Trichoderma harzianum was applied to cucumber and pepper seedlings as a peat-bran preparation incorporated into the propagative mixture in a commercial production nursery. On marketing day (after 18 and 30 days for cucumber and pepper, respectively), significant increases of 23.8% and 17.2% in seedling height, 96.1% and 50% in leaf area, and 24.7% and 28.6% in plant dry weight were observed in cucumber and pepper seedlings, respectively, as compared to their non-treated counterparts.Trichoderma-treated seedlings were much more developed and vigorous and had higher chlorophyll contents. No significant differences were found in N, P or K content between treatments. Cucumber seedlings were then transplanted to a commercial greenhouse and analyzed over two successive growth cycles following soil fumigation with methyl bromide (500 kg/ha). Results revealed theTrichoderma-treated plants to be more resistant to damping-off disease. During the first cycle, immediately after soil fumigation, no damping-off was observed with either treatment, except in border beds where 4% of the non-treated plants died, as compared to no damping-off in theTrichoderma-treated plants. During the second growing cycle however, significant reductions in damping-off of 67% and 52% were obtained in middle and border beds, respectively, as compared to the non-treated controls.     

Biochemical defense responses of black pepper (Piper nigrum L.) lines to Phytophthora capsici

A study on biochemical factors involved in black pepper defense response against Phytophthora capsici, was carried out in P. capsici susceptible (Sreekara) and resistant (04-P24, shows root resistance to the pathogen) black pepper lines. Seven important factors – change in membrane conductance, total phenols, orthodihydroxy (OD) phenols, lignin and defense related enzymes (peroxidase, β-1,3 glucanase and β-1,4 glucanase) – were studied under uninoculated and pathogen (P. capsici, isolate 06-04) inoculated condition to know the preformed and induced responses. The pathogen was inoculated (soil inoculation) and plants were observed for changes, at 24 h intervals for 10 days. On 8th day after inoculation symptoms started appearing on Sreekara and increased the severity till 10th day. Both root and stem samples were subjected for biochemical analysis. Of the factors analyzed, it was found that membrane conductance, OD phenol, lignin and peroxidase activity play significant role in root resistance to P. capsici in 04-P24. Light microscopy of the portion of root – where pathogen found attached – was also done.     

In vivo control and in vitro antifungal activity of rhamnolipid B,a glycolipid antibiotic,against Phytophthora capsici and Colletotrichum orbiculare

The glycolipid antibiotic rhamnolipid B isolated from Pseudomonas aeruginosa strain B5 was evaluated for in vitro antifungal activity and in vivo control against phytophthora blight and anthracnose under glasshouse conditions. Rhamnolipid B showed antifungal activity against Cercospora kikuchii, Cladosporium cucumerinum, Colletotrichum orbiculare, Cylindrocarpon destructans, Magnaporthe grisea and Phytophthora capsici. Microscopic observation revealed that the high level of antifungal activity (10 µg ml ⁻¹) against P capsici was mainly due to a lytic effect on zoospores. Zoospore lysis began in the presence of 10 µg ml ⁻¹ of rhamnolipid B and most of the zoospores were collapsed at 25 µg ml ⁻¹. Rhamnolipid B showed inhibitory activity against the germination of zoospores and hyphal growth of P capsici at concentrations of 50 µg ml ⁻¹. Spore germination of the anthracnose plant pathogen C orbiculare was also inhibited in the presence of 50 µg ml ⁻¹ of rhamnolipid B, although hyphal growth was not affected at this concentration. In the glasshouse, the efficacy of rhamnolipid B against phytophthora blight was similar to that of metalaxyl on pepper plants when treated just before inoculation with P capsici. Treatment with either at 500 µg ml ⁻¹ completely protected pepper plants from phytophthora blight. Rhamnolipid B also suppressed the development of C orbiculare infection on leaves of cucumber plants. © 2000 Society of Chemical Industry     

Trichoderma harzianum T39 and T. virens DAR 74290 as Potential Biological Control Agents for Phytophthora erythroseptica

Trichoderma harzianum isolate T39 and T. virens isolate DAR 74290 were evaluated as potential biological agents for control of pink rot of potato and root and stem rot of tomato caused by Phytophthora erythroseptica. Cell-free metabolites of T. virens DAR 74290 completely inhibited growth of P. erythroseptica in vitro and appeared to be fungicidal. T. virens DAR 74290 and Trichodex, a commercial formulation of T. harzianum T39, were tested for their ability to protect potato and tomato plants from disease caused by P. erythroseptica in glasshouse experiments. Trichodex and T. virens DAR 74290, alone and combined, reduced disease severity in shoots and roots of potatoes 10 weeks after inoculation with the pathogen. The yield of potatoes from plants treated with P. erythroseptica and T. virens DAR 74290 (mean of 12.9g fresh weight/pot) was significantly greater than in controls inoculated with the pathogen alone (mean of 2.1g/pot). Treatment with Trichodex alone increased the yield of tubers compared to the uninoculated controls. T. virens DAR 74290 increased the survival of tomato seedlings inoculated with the pathogen, and both this isolate and Trichodex decreased the severity of disease on tomato.     

Biological control ofBotrytis cinerea on tomato stem wounds withTrichoderma harzianum

The effectiveness ofTrichoderma harzianum in suppression of tomato stem rot caused byBotrytis cinerea was examined on tomato stem pieces and on whole plants. Ten days after simultanous inoculation withB. cinerea andT. harzianum, the incidence of infected stem pieces was reduced by 62–84%, the severity of infection by 68–71% and the intensity of sporulation by 87%. Seventeen days after inoculation of wounds on whole plants, the incidence of stem rot was reduced by 50 and 33% at 15 and 26 °C, respectively, and the incidence of rot at leaf scar sites on the main stem was reduced by 60 and 50%, respectively. Simultanous inoculation and pre-inoculation withT. harzianum gave good control ofB. cinerea (50 and 90% disease reduction, 10 days after inoculation). The rate of rotting was not reduced by the biocontrol agent once infection was established. However, sporulation byB. cinerea was specifically reduced on these rotting stem pieces. Temperature had a greater effect than vapour pressure deficit (VPD) on the efficacy of biocontrol. Suppression ofB. cinerea incidence byT. harzianum on stem pieces was significant at 10 °C and higher temperatures up to 26 °C. Control of infection was significantly lower at a VPD of 1.3 kPa (60% reduction), than at VPD<1.06 kPa (90–100% control). Reductions in the severity of stem rotting and the sporulation intensity of grey mould were generally not affected by VPD in the range 0.59–1.06 kPa. Survival ofT. harzianum on stems was affected by both temperature and VPD and was greatest at 10 °C at a low VPD and at 26 ° C at a high VPD.     

Histological and ultrastructural comparisons of compatible, incompatible and -β-amino- n -butyric acid-induced resistance responses of pepper stems to Phytophthora capsici

The compatible interaction of pepper stems with Phytophthora capsici showed more rapid and severe disease development than did the incompatible interaction, although pathogen penetration styles of host cells in compatible and incompatible interactions were similar to each other. Treatment with -β-amino- n -butyric acid (BABA) protected the pepper plants against P. capsici infection. Reduced hyphal growth and sporangial formation were found after P. capsici infection in BABA-induced resistant and incompatible reactions. One of the most noticeable ultrastructural features of the BABA-induced resistant reaction was the formation of electron-dense wall appositions. The thick and dense wall appositions that encased the haustoria restricted haustorial development, thus leading to limitation of further pathogen penetration into inner plant tissues. A main host response in the incompatible interaction was the occlusion of cortical cells with an amorphous material. Plugging of the intercellular spaces in the cortical cells with electron opaque material was frequently observed in the incompatible interaction, but not in the compatible interaction. Another common feature of the BABA-induced resistant and incompatible reactions was degeneration of mitochondrial structure within penetrating hyphal cytoplasm. The mitochondrial structure in the BABA-induced resistant or incompatible reactions had no distinct double membrane layer and well-shaped cristae.     

Phytophthora root rot of sweet pepper

Phytophthora capsici proved to be the causal agent of a root and crown rot of sweet pepper in the Netherlands.P. capsici was pathogenic on sweet pepper, tomato and sometimes on eggplant but not on tobacco Xanthi. Of these test plants only tomato was infected byP. nicotianae.No different symptoms in plants infected with eitherP. capsici orP. nicotianae were found. Dipping the roots of tomato and sweet pepper plants in a suspension ofP. capsici resulted in a more severe attack than pouring the suspension on the stem base.Resistance in tomato toP. nicotianae did not include resistance toP. capsici. A method to distinguishP. capsici fromP. nicotianae after isolation from soil is described. Both species were able to infect green fruits of tomato and sweet pepper.p. capsici survived in moist soil in the absence of a host for at least 15 months.Samenvatting Phytophthora capsici bleek de oorzaak te zijn van een voet-en wortelrot in paprika op twee bedrijven in 1977 in Nederland.P. capsici was pathogeen op paprika, tomaat en soms op aubergine maar niet op tabak Xanthi.P. nicotianae tastte van deze toetsplanten alleen tomaat aan. Verschillen in symptomen tussenP. nicotianae enP. capsici werden bij tomaat niet waargenomen.Het dompelen van de wortels in eenP. capsici suspensie gaf een ernstiger aantasting dan het begieten van de wortelhals met deze suspensie.Resistentie in tomaat tegenP. nicotianae bleek geen resistentie tegenP. capsici in te houden. P. capsici kan in grond worden aangetoond door groene paprikavruchten als vangsubstraat te gebruiken.P. capsici enP. nicotianae kunnen beide zowel vruchten van tomaat als paprika aantasten. P. capsici overleefde een periode van 15 maan den in vochtige grond waarop geen waardplant werd geteeld.     

Interactive Effects of Two Soilborne Pathogens, Phytophthora capsici and Verticillium dahliae, on Chile Pepper

ABSTRACT Phytophthora capsici and Verticillium dahliae are two mycelial microorganisms associated with wilt symptoms on chile pepper (Capsicum annuum). Both pathogens occur in the same field and can infect a single plant. This study examined the nature of the co-occurrence of P. capsici and V. dahliae. Chile pepper plants were inoculated with each pathogen separately or with both pathogens concomitantly or sequentially. In concomitant inoculations, plants were inoculated with a mixture of zoospores of P. capsici and conidia of V. dahliae. In sequential inoculations, plants were inoculated with zoospores of P. capsici 4 days prior to inoculation with conidia of V. dahliae, or plants were inoculated with conidia of V. dahliae 4 days prior to inoculation with zoospores of P. capsici. Stem necrosis and leaf wilting were visible 3 to 4 days earlier in plants inoculated with both P. capsici and V. dahliae than in plants inoculated with P. capsici alone. Stem necrosis and generalized plant wilting were observed in plants inoculated with P. capsici alone, and stem necrosis, generalized plant wilting, and vascular discoloration were observed in plants inoculated with both P. capsici and V. dahliae by 21 days after inoculation. These symptoms were not observed in control plants or plants inoculated with V. dahliae alone. The frequency of recovery of V. dahliae from stems was approximately 85 to 140% higher across inoculum levels when plants were inoculated with both P. capsici and V. dahliae than when plants were inoculated by V. dahliae alone. Similarly, the frequency of recovery of V. dahliae from roots was approximately 13 to 40% higher across inoculum levels when plants were inoculated with both P. capsici and V. dahliae than when plants were inoculated by V. dahliae alone. There was no apparent antagonism between the two pathogens when they were paired on growth media. In general, when P. capsici and V. dahliae were paired on growth media, mycelial growth of each pathogen grown alone was not significantly different from mycelial growth when the pathogens were paired. Results suggest that wilt development is hastened by the presence of both P. capsici and V. dahliae in the same plants. The presence of P. capsici and V. dahliae in the same inoculum court enhanced infection and colonization of chile pepper by V. dahliae.     

Extracellular self-DNA plays a role as a damage-associated molecular pattern (DAMP) delaying zoospore germination rate and inducing stress-related responses in Phytophthora capsici

Phytophthora capsici is a highly destructive pathogen of crops. Although chemical pesticides are the most widely used strategy to counter phytopathogens, they have been inefficient to combat P. capsici and have produced significant environmental and health problems. Therefore, sustainable alternatives to control soilborne pathogens, such as the inhibitory effect of self-extracellular DNA (eDNA), have been proposed. This inhibition phenomenon has been attributed to the action of self-eDNA as a damage-associated molecular pattern (DAMP). Here, we describe the effect of self-eDNA on P. capsici zoospore germination rate, antioxidant enzymes activity and MAPK gene expression. Also, the effect of P. capsici eDNA on the protection of chilli pepper (Capsicum annuum) plants against P. capsici was investigated. The results highlight that P. capsici can sense 2–500 µg/ml self-eDNA and induce stress-related responses like SAK1 gene expression, and superoxide dismutase and catalase activities. Moreover, in vitro zoospore germination rate was suppressed with self-eDNA concentrations ranging from 50 to 500 µg/ml. Interestingly, drench applications of P. capsici eDNA at 60 and 100 µg/ml on chilli pepper plants did not show any protective effect against the phytopathogen, whereas 2 µg/ml of P. capsici eDNA drench application showed a lower percentage of plants with symptoms and lower disease severity. Moreover, phenols and total flavonoids were increased in chilli pepper plants, therefore inducing plant immunity. This study showed that self-eDNA acts as a DAMP in P. capsici and provides insight into the use of eDNA for the protection of crops of agronomic interest.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.     

哈茨木霉菌对水稻幼苗根际土壤微生物和酶活性的影响

采用钵盘育苗试验,研究了哈茨木霉菌在调节水稻苗床土壤微生物群落及土壤酶活性中的作用。研究结果表明：哈茨木霉菌能有效调节水稻幼苗根际土壤微生物群落组成;哈茨木霉菌对细菌、真菌和放线菌数量的影响程度明显不同,播种28天后,哈茨木霉菌接种处理的水稻幼苗根际土壤细菌和放线菌数量较对照分别增加50.70%和48.56%,而真菌数量较对照减少16.15%,并且哈茨木霉菌数量较刚播种时增加了138.46%;哈茨木霉菌也能显著提高土壤脲酶、磷酸酶和蔗糖酶的活性,分别较对照提高8.55%、18.31%和49.61%。研究表明,哈茨木霉菌具有改善土壤微生态环境的作用。