Combined use of Streptomyces sp. A6 and chemical fungicides against fusarium wilt of Cajanus cajan may reduce the dosage of fungicides required in the field

Biological control agents offer one of the best alternatives to reduce the use of pesticides. This investigation studied the tolerance to fungicides and integrated use of the potential biocontrol agent Streptomyces sp. A6 for control of Fusarium wilt of pigeon pea, Cajanus cajan. Streptomyces sp. A6 exhibited strong tolerance towards most of the fungicides used in the study at concentrations higher than those recommended for field applications. The isolate showed enhanced growth and mycolytic enzyme production in the presence of sulphur, mancozeb, carbendazim, fosetyl aluminium and triadimefon. The fungicides mancozeb, sulphur and carbendazim were selected for further studies. Effective concentrations (EC₅₀ values) of the test fungicides that reduced Fusarium spore germination and fungal biomass by 50% were determined. Similarly, the EC₅₀ for inhibiting fungal spore germination and reducing fungal biomass to 50% by Streptomyces sp. A6 and culture filtrate (CF) were also determined. Combining the EC₅₀ dose of the culture and CF with test fungicides was found to be more effective for controlling Fusarium infection in C. cajan compared to the sum of the effects of the individual treatments. Such combined use of biocontrol agent with fungicides can reduce the dosage of toxic fungicides in agricultural fields, thereby reducing environmental risks. Tolerance and synergistic interaction of Streptomyces sp. A6 with frequently used fungicides suggested its potential in integrated pest management. To the best our knowledge, this is the first extensive study on integrated use of Streptomyces species with fungicides.     

Wilt disease management and enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations amended with chemical fertilizer

Root nodulating Sinorhizobium fredii KCC5 and Pseudomonas fluorescens LPK2 were isolated from nodules of Cajanus cajan and disease suppressive soil of tomato rhizosphere, respectively. Both strains produced IAA, siderophore, solubilized insoluble phosphate, showed chitinase and β-1,3-glucanase activities, and strongly inhibited the growth of Fusarium udum. It also caused degradation and digestion of cell wall components, resulting in hyphal perforations, empty cell (halo) formation, shrinking and lysis of fungal mycelia along with significant degeneration of conidia. LPK2 produced volatile cyanogen (HCN). Combinations of S. fredii KCC5 and P. fluorescens LPK2 with half dose of chemical fertilizers showed a significant increase in seed germination (94%) while seed germination with co-inoculated strains (KCC5 + LPK2), KCC5 and LPK2 alone was 90, 84 and 82% respectively as compared to control 77%. After 120 days of sowing, per plant number of pods, nodules, shoot length, root length, shoot weight and root weight were greater for the combination with half dose of chemical fertilizers compared to the control. Combinations of S. fredii KCC5 and P. fluorescens LPK2 with half dose of chemical fertilizers resulted in an 82% increase in grain yield per hectare compared to the control. Both strains KCC5 and LPK2 led to proto-cooperation as evidenced by synergism, aggressive colonization of the roots, and enhanced growth, suggesting potential biocontrol efficacy against Fusarium wilt in C. cajan.     

Effects of fungicide seed treatments on germination, population, and yield of maize grown from seed infected with fungal pathogens

Seedborne fungi can reduce survival, growth, and yield of maize (Zea mays L.). Laboratory, field, and growth chamber experiments were conducted to determine the effects of the seed treatment fungicides fludioxonil, mefenoxam, and azoxystrobin on germination, plant population, and grain yield of maize grown from low-quality hybrid seed infected with seedborne fungal pathogens. Study I used seed of four hybrids infected at 0-54% incidence with Fusarium spp., Stenocarpella maydis, Penicillium spp., Rhizopus spp., and/or Aspergillus spp. Study II used three seed lots for each of two hybrids infected at 7-37% incidence with S. maydis. Warm and cold germination for untreated seed varied among hybrids in both studies. Warm germination of the seed lot with the highest incidence of S. maydis in study II treated with azoxystrobin and fludioxonil was significantly greater (+7%) than the nontreated control. Plant population in study I was significantly affected by seed treatment, hybrid, and their interactions. Populations were greater (≥9%) for fludioxonil, fludioxonil + mefenoxam, and fludioxonil + mefenoxam + azoxystrobin treatments compared to controls. In growth chamber experiments with pasteurized soil, emergence (≥5%) and plant dry weight (≥14%) were both greater than controls only with the triple seed treatment. Plant populations in study II for all seed treatments except mefenoxam and azoxystrobin alone were greater (≥4%) than controls. Yield in study I was significantly affected by hybrid and seed treatment. Yield for one hybrid was higher (≥20%) than the control with all seed treatments except fludioxonil, whereas yield with another hybrid was consistently greater (≥26%) only with the triple seed treatment. Yield in study II was significantly affected by hybrid, seed treatment, and their interactions. Yield was greater (≥8%) than the controls for all seed treatments with one hybrid and with all (≥5%) except azoxystrobin for the other hybrid. Highest yields occurred with the triple seed treatment. Results indicate that fludioxonil and azoxystrobin can increase germination, population, and yield of maize grown from seed infected by S. maydis and other fungi.     

Genotype-by-environment interaction of barley DH lines infected with Fusarium culmorum (W.G.Sm.) Sacc.

Infection of plants by pathogens is a biotic environmental stress. Barley plants are infected, among others, by Fusarium culmorum—a pathogen affecting seedling, head, root and stem. The infection can result in reduced yield and grain quality. The aim of the study was to compare the reaction of inoculated and non-inoculated barley doubled haploids (DHs) with F. culmorum in various environments. Thirty-four genotypes were inoculated with an isolate of F. culmorum. The experiment was carried out over 6 years. Kernel weight per spike, 1000-kernel weight and percentage of plump kernels were observed in control and inoculated plants. Genotype-by-environment (GE) interaction and its structure with reference to the environments and genotypes were analysed. Additional information about the sensitivity of healthy and infected genotypes to environments was determined by the regression analysis. Statistical computation was made using the SERGEN software. Lines were considered as unstable when their GE interaction was significant at P = 0.05. Unstable genotypes were classified as intensive or extensive according to the results of the regression analysis. It was found that infection with Fusarium decreased the stability of barley lines in different environments. Interaction of unstable infected genotypes with environments, most often, could not be explained by the regression—their response to various environmental conditions appeared to be unpredictable. Selection of lines less susceptible to biotic and abiotic stresses was possible due to comparison of classification of healthy and infected lines, which was made based on their main effects and GE interaction.     

Biocontrol of Rhizoctonia solani and Sclerotium rolfsii on tomato by delivering antagonistic bacteria through a drip irrigation system

In a 2-year assessment carried out on tomato crops of Central and Southern Italy, a high incidence of Rhizoctonia solani and Sclerotium rolfsii was found. These fungal pathogens attack horticultural crops and are responsible for severe crown and stem rot. Because of technical, economical and environmental issues their chemical control is an arduous task. To find alternative and eco-compatible control methods, the effectiveness of two new antagonistic bacterial isolates (Burkholderia cepacia, T1A-2B, and Pseudomonas sp., T4B-2A), previously selected from suppressive organic amendments, were tested on tomato plants grown under both growth chamber and field conditions. The potential antagonists were compared with two commercial biofungicides, based on Bacillus subtilis (BSF4) and Trichoderma asperellum (TV1), and four synthetic fungicides (tolclofos-methyl, azoxystrobin, fosetyl-Al and fosetyl-Al + propamocarb). In 2-year field experiments carried out on tomato plants, the biocontrol bacteria as well as the other treatments were applied to the soil, proximal to the plant crowns and main roots, by means of an effective and specific system of drip irrigation. In all the experiments the novel selected biocontrol bacteria significantly reduced both incidence and severity of the diseases caused by S. rolfsii or R. solani, with results demonstrating effectiveness equal to TV1, better than BSF4 and comparable with the synthetic fungicides, except for tolclofos-methyl which was the most effective treatment. In field experiments, carried out for two consecutive years, isolate T1A-2B reduced up to 58.33% and up to 63.8% the severity of the diseases caused by S. rolfsii and R. solani respectively; whereas isolate T4B-2A gave reduction of S. rolfsii and R. solani diseases severity up to 73.2% and up to 62.7%, respectively.     

Management of the root-knot nematode Meloidogyne incognita on tomato with combinations of different biocontrol organisms

The nematicidal effect of Pseudomonas fluorescens, Paecilomyces lilacinus, Pichia guilliermondii and Calothrix parietina singly or in combination was tested against root-knot nematode, Meloidogyne incognita. Treatments with P. fluorescens and P. lilacinus caused mortality of M. incognita as 45% and 30% of juveniles after 48 h of exposures, respectively compared to water control in vitro. Under greenhouse conditions, all treatments reduced the disease severity and enhanced plant growth compared to untreated control. Application of P. fluorescens, P. lilacinus and P. guilliermondii Moh 10 was more effective compared to C. parietina. There was a negative interaction between C. parietina and either P. lilacinus or P. guilliermondii. Fresh and dry weight of shoots and roots of plants were significantly reduced as a result of infection with M. incognita, however application of biocontrol agents singly or in mix recovered this reduction. Moreover, they enhanced the growth parameters compared with the control. Our results proved that application of different biocontrol agents (P. fluorescens, P. lilacinus and P. guilliermondii) not only has a lethal effect on nematode, but also enhances the plant growth, supplying many nutritional elements and induction the systemic resistance in plants. Presence of C. parietina as a soil inhabitant cyanobacterium could antagonize biocontrol agents leading to the reduction of their practical efficiency in soil.     

Effect of Trichoderma asperellum and arbuscular mycorrhizal fungi on cacao growth and resistance against black pod disease

Arbuscular mycorrhizal fungi (AMF) and members of the genus Trichoderma have emerged as promising groups of microbial inoculants that can induce plant growth and resistance to disease. This study aimed at investigating the potential of AMF and a strain (PR11) of Trichoderma asperellum to promote cacao growth and induce resistance against Phytophthora megakarya. Cacao seedlings were either non-inoculated, or inoculated with the saprophytic fungus T. asperellum and/or a mixture of two different mycorrhizal fungi, Gigaspora margarita and Acaulospora tuberculata. Eighteen weeks after planting, a series of morphological as well as biochemical changes, which are considered to be part of the plant defense response, were measured after a challenge inoculation of the leaves with zoospores of P. megakarya. Inoculation with AMF and T. asperellum alone was essential for the promotion of plant growth. Significant increase in plant height, root and shoot fresh weights, as well as phosphorous uptake was recorded in comparison to non-inoculated control plants. However, dual inoculation of cacao seedlings with T. asperellum and AMF did not always positively benefit the plants. Leaf inoculation showed variation among the treatments, with the lowest disease index (highest level of resistance) recorded in plants inoculated with either AMF or T. asperellum only. This came along with a high synthesis of amino acids and phenolic compounds in both healthy and infected leaves, suggesting that these metabolites are implicated in disease resistance.     

Effectiveness of plant extracts on suppression of damping-off and wilt diseases of lupine (Lupinus termis Forsik)

The aim of this study was to evaluate water and organic solvent of plant extracts for protection of lupine plants against damping-off and wilt diseases caused by Fusarium oxysporum f. sp. lupini, F. oxysporum f. sp. lupini Snyder & Hansen was isolated from diseased lupine roots collected from different locations of Minia, Assiut and New Valley governorates. Water leaf extracts of Calotropis procera, Nerium oleander, Eugenia jambolana, Citrullus colocynthis, Ambrosia maritime, Acacia nilotica and Ocimum basilicum and fruit extracts of C. colocynthis, C. procera and E. jambolana reduced damping-off and wilt diseases caused by F. oxysporum f. sp. lupini. Water extracts of E. jambolana leaves, C. colocynthis fruits and N. oleander leaves were the most effective ones for controlling such diseases. In contrast, A. nilotica and O. basilicum extracts were the least effective ones. Organic solvent extracts of tested plants reduced damping-off and wilt diseases than water extracts. Butanolic and ethereal extracts were highly effective in reduction of diseases than the other tested extracts. Under field conditions, during winter growing seasons 2008/09 and 2009/10, ethereal and butanolic extracts of N. oleander and E. jambolana leaves and C. colocynthis fruits significantly reduced the percentage of wilt severity as well as improved growth parameters (plant height, number of branches, pods and seeds per plant) and increased seed index, total seed yield/hectare compared with control treatment, while protein content in seeds was not effected. In conclusion, our study demonstrated that some plant extracts can be used for biocontrol of lupine damping-off and wilt diseases.     

Actinomycetes mediated biochemical responses in tomato (Solanum lycopersicum) enhances bioprotection against Rhizoctonia solani

Six actinomycetes isolates, namely Streptomyces toxytricini vh6, Streptomyces flavotricini vh8, S. toxytricini vh22, Streptomyces avidinii vh32, Streptomyces tricolor vh85 and vh41, an isolate of an unknown species of Actinomycetales, were tested for their efficacy in protecting tomato (Solanum lycopersicum) against Rhizoctonia solani under green house conditions. Actinomycetes treated plants showed better growth in terms of high chlorophyll content, higher phenylalanine ammonia lyase (PAL) activity and high total phenolic content. Qualitative and quantitative estimation of phenolic compounds from tomato leaves showed significant accumulation of six phenolic acids, gallic (29.02 μg g⁻¹ fresh leaf wt), ferulic (11.44 μg g⁻¹ fresh wt), cinnamic (56.84 μg g⁻¹ fresh wt), gentisic (24.19 μg g⁻¹ fresh wt), chlorogenic acid (1.72 μg g⁻¹ fresh wt) and salicylic (0.39 μg g⁻¹ fresh wt) acid, in actinomycetes treated plants. Biochemical profiling, when correlated with plant mortality in actinomycetes treated and untreated plants, indicated that isolates vh6 and vh8 offered 44.55% and 40.14% disease reductions, respectively compared to the control. These results established that these organisms have the potential to act as biocontrol agents.     

Effects of crop rotation of soybean with corn on severity of sudden death syndrome and population densities of Heterodera glycines in naturally infested soil

Sudden death syndrome, caused by Fusarium virguliforme, and the soybean cyst nematode, Heterodera glycines, combined cause the highest yield losses in soybean. The objective of this study was to determine the effectiveness of corn rotated annually with soybean on reducing severity of sudden death syndrome (SDS) and if such crop rotation is beneficial to soybean root health and thus improves disease management strategies. Experiments were conducted from 2003 to 2006 through two cycles of a corn–soybean rotation on two commercial fields in Indiana. With one exception, the rotation of soybean with corn did not provide yield benefits compared to monoculture of soybean. Severity of foliar and root symptoms of SDS in rotation plots were never less than in soybean monoculture plots. At one location, soybean monoculture resulted in suppression of SDS compared to the corn–soybean rotation, while H. glycines reproduced freely. At the other location, monoculture of soybean resulted in suppressiveness against H. glycines, while SDS was limited in all treatments. The data suggest that soil suppressiveness can independently impact the pathogens that are important in SDS development. Because H. glycines can increase SDS symptoms, its suppression may also reduce severity of SDS. Current production systems consisting of yearly rotation of soybean with corn are highly vulnerable to the development of severe soil-borne disease complexes. The simple year-to-year rotation of corn and soybean is not considered sustainable. While monoculture of soybean resulted in some disease suppression in these trials, reliance on monoculture may be detrimental due to other environmental considerations beyond the scope of these trials. Including other crops may be beneficial in improving the sustainability of soybean and corn production systems.     

In vitro activity of eighteen essential oils and some major components against common postharvest fungal pathogens of fruit

The development of natural crop protective products as alternatives to synthetic fungicides is currently in the spotlight. In vitro experiments are valuable precursors to more costly in vivo trials, allowing the identification of effective essential oils and establishing the concentrations required for inhibition of a specific, or spectrum of decay pathogens. In this study, the antifungal properties of eighteen essential oils were evaluated in vitro by addition to the fungal growth medium of five pathogens (Lasiodiplodia theobromae, Colletotrichum gloeosporioides, Alternaria citrii, Botrytis cinerea and Penicillium digitatum) isolated from mango, avocado, citrus, grapes and cactus pear. The inhibitory properties of some of the major compounds of the oils, identified and quantified by gas chromatography-mass spectroscopy and gas chromatography-flame ionization detection were also determined. Most of the oils were selected on the basis of commercial availability and for containing a predominant compound. Visual inspection of fungal growth was done and the lowest concentration where fungal growth was completely inhibited on all replicates was recorded. Thyme oil proved to be the most effective inhibitor, totally inhibiting all of the pathogens tested at concentrations of 1000 μl/l and lower, with the exception of a resistant Penicillium strain. Cinnamon oil, rich in eugenol (81.2%), demonstrated good fungicide potential, while the carvone-rich oils displayed promising activity against the citrus pathogens. Oils characterized by high concentrations of S-carvone were less effective than those containing the R-enantiomer. Essential oil of Lippia citriodora was active against all of the pathogens, excluding L. theobromae from avocado. These essential oils, applied alone or in combination, are good candidates for further in vivo testing and for investigations concerning their modes of action.     

Field evaluation of yield effects on the U.S.A. heirloom sweetpotato cultivars infected by Sweet potato leaf curl virus

The incidence of Sweet potato leaf curl virus (SPLCV), a Begomovirus, on sweetpotato, Ipomoea batatas (L.) Lam. (Convolvulaceae), in South Carolina, U.S.A. has increased rapidly in recent years. This is likely due to the use of infected propagating materials and the increasing population of its vector, the sweetpotato whitefly, Bemisia tabaci (Gennadius). In this study, we demonstrated in field experiments that SPLCV infection reduced the yields of most heirloom sweetpotato lines relative to the yields of non-infected plants. Real-time polymerase chain reaction (PCR) technology specific to several common sweetpotato viruses was used to determine the virus infection status in 69 selected accessions of heirloom sweetpotato lines. Meristem tip culture technology was used to regenerate virus-free plants from these materials. To ensure the virus-free status, each mericlone was evaluated using real-time PCR and graft bio-indexing on the indicator species, Ipomoea setosa Ker Gawl. Mericlones of 27 cultivars were found to be free of the viruses. The 27 cultivars were included in a field test to determine the effect of SPLCV infection on yield. Yields of virus-free plants of the cultivars ranged from 10 to 80% greater than the yields of SPLCV-infected plants. However, the yield differences between virus-free and infected plants were diminished in the second year of the field experiment due to a rapid re-infection by SPLCV. These results demonstrate the importance of using certified, virus-tested seed roots or cuttings. The rapid re-infection of the virus-tested sweetpotato plants with SPLCV observed in these studies suggests that management of the whitefly population should be a critical element in control of this important virus.     

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.     

Visual inspections of nursery stock fail to protect new plantings from Blueberry scorch virus infection

Blueberry scorch virus (BlScV) is one of the most pervasive pathogens of highbush blueberry. The virus is aphid-vectored and exhibits a latent period between infection and symptom expression in the host plant of up to 5 years. In many cases, we have observed BlScV symptom expression in new fields that appears inconsistent with aphid-vectored introduction and spread. It was, therefore, speculated that the virus may be introduced through infected nursery stock. To examine this possibility, we first surveyed selected nurseries to determine if mother plants, used for propagation by cuttings, were BlScV-infected. Two nurseries were found to harbor symptomless, infected, mother plants (cv. Duke). Cuttings from one nursery were collected from infected and non-infected plants and rooted in propagation beds. The survival and infection of cohorts from each mother plant were determined one year after planting. A significantly greater proportion of cuttings survived from non-infected mother plants (0.7) than from infected mother plants (0.5). Of the cohort from infected mother plants that survived, 40% tested positive for BlScV. We also surveyed the distribution of infected, symptomatic plants in recent ‘Duke’ plantings that originated from nurseries with BlScV-infected mother plants and compared distribution with older plantings with more advanced BlScV outbreaks. In all cases, the distribution of BlScV symptom development in young fields was random, which is consistent with introduction from planting stock. Older plantings showed a strong clustered distribution, which is consistent with aphid transmission. This study identifies infected nursery stock as an important source of BlScV dissemination and underscores the importance of having symptomless mother plants virus tested.     

Fusarium genetic traceability: Role for mycotoxin control in small grain cereals agro-food chains

Risks associated with mycotoxin contamination of cereals, that are included in the ten major staple foods and greatly contribute to the dietary energy intake, are of worldwide relevance. In small grain cereals, mycotoxins are produced by fungi such as Aspergillus, Penicillium, Alternaria and Fusarium that colonize the plant in the field and can grow during the post-harvest period, producing several classes of mycotoxins. The identification of mycotoxigenic fungal species and strains is essential for developing effective strategies for control. For this purpose, genetic traceability has proved to be a valuable tool that can be applied along the whole production chain, starting in the field for early diagnosis of FHB (Fusarium Head Blight) disease to the final processing steps, such as malting or pasta making. In this paper, DNA-based analytical tools that are currently available for the identification and quantification of mycotoxigenic fungal species and strains are reviewed, with particular emphasis on Fusarium, and their possible applications in mycotoxin control in small grain cereal chains are discussed.     

Disinfection using ozone microbubbles to inactivate Fusarium oxysporum f. sp. melonis and Pectobacterium carotovorum subsp. carotovorum

To establish a method for disinfecting hydroponic culture solutions using ozone microbubbles (OMB), we examined the disinfectant activity of OMB against Fusarium oxysporum f. sp. melonis and Pectobacterium carotovorum subsp. carotovorum in infected plant roots. OMB had a higher solubility and remained in the water for a longer period than ozone millibubbles, resulting in extremely high disinfecting activity against both phytopathogens. Furthermore, disinfectant activity and durability of OMB-treated water against both phytopathogens increased with an increase in the initial concentration of dissolved ozone. Therefore, these results suggest that OMB may be suitable for use as a new disinfectant against phytopathogens in hydroponic culture solutions.     

Study on antagonistic effects of Talaromyces flavus on Verticillium albo-atrum, the causal agent of potato wilt disease

Talaromyces flavus a fungal antagonist, was isolated from soil samples collected from potato fields in Varamin and Karaj districts, Tehran province, Iran. Antagonistic effects of T. flavus isolates against Verticillium albo-atrum, the causal agent of potato wilt disease were investigated in the laboratory and greenhouse conditions. T. flavus colonies were recovered after three weeks from soil samples cultured on selective medium. Antagonistic effects of volatile and non-volatile extracts of T. flavus isolates on V. albo-atrum growth were investigated in the laboratory and five that caused higher growth inhibition of V. albo-atrum, were selected for greenhouse experiments. Infection index was compared in the greenhouse in a split plot trial with five isolates applied to soil, seed, or both arranged in a randomized complete block design with four replications. The minimum infection index was observed when seed were treated with T. flavus with the most effective isolate being Tf-Po-V-52. On seed, the minimum infection index was observed with Tf-Po-V-50. The most effective T. flavus isolate was also evaluated in a field experiment. Results indicated that infection index in seed treatment contained this isolate was 0.15 whereas that of control was 3.5. The overall results of this study showed that it may be possible to manage potato Verticillium wilt disease effectively by using T. flavus, a biocontrol fungus.     

Growth promotion and induction of resistance in tomato plant against Xanthomonas euvesicatoria and Alternaria solani by Trichoderma spp.

In tomato crop, the induction of resistance emerges as an important alternative for achieving the reduction of chemicals in disease control. This study aimed to evaluate the ability of 28 Trichoderma isolates to promote the growth of tomato seedlings and to induce systemic resistance (ISR) against Xanthomonas euvesicatoria and Alternaria solani, the causal agents of bacterial spot and early blight, respectively. Twelve isolates promoted the increase of plant dry matter mass (DMM) above 100%, showing the great potential of these strains. All isolates were able to colonize the root system of tomato plants. The plant growth-promoting isolates were further evaluated for potential elicitation of ISR. Treatment of the soil with all Trichoderma isolates provided protection in tomato plants from 24.13 to 95.94% against X. euvesicatoria and 30.69 to 95.23% against A. solani. The most efficient isolates in reducing the severity of bacterial spot and early blight were the isolates IB 28/07, IB 30/07, IB 37/01 and IB 28/07, IB 30/07 and IB 42/03, respectively. The effect of different time intervals between Trichoderma application and inoculation with pathogens in inducing systemic resistance in tomato plants was evaluated for the isolate IB 28/07. IB 28/07 conferred protection against both diseases at all time intervals, confirming the ability of the isolate to reduce the severity of these diseases up to 21 days after treatment of tomato plants. In vitro assays revealed that all isolates of Trichoderma were able to degrade cellulose. Only the isolate IB 34/08 showed antagonistic activity against X. euvesicatoria and none caused reduction in the in vitro mycelial growth of A. solani. Trichoderma isolates were identified at species level by DNA sequencing.     

Growth and accumulation of caffeic acid derivatives in Echinacea angustifolia DC. var. angustifolia grown in hydroponic culture

In separate experiments conducted in 2007 and 2008, growth and accumulation of selected caffeic acid derivatives (CADs; i.e., caftaric acid, chlorogenic acid, echinacoside, caffeic acid, cynarin, p-coumaric acid, ferulic acid and cichoric acid) were determined in Echinacea angustifolia DC. var. angustifolia seedlings grown in hydroponic culture (floating raft system) at a density of 122 plant m⁻² (at planting). Plants were harvested 11 (2007) or 16 (2008) weeks after transplanting (i.e., 15 or 20 weeks after sowing). In both years, plants grew vigorously and at harvest approximately half of the plants under observation had developed one to three inflorescences. In 2008, the root yield (2940 kg ha⁻¹) harvested in nearly eight months from two consecutive hydroponic cultures was within the yield reported in the literature for field cultivations lasting two to four years. None of the selected CADs was found in the leaves, while the inflorescences (stem and capitulum) contained only caftaric acid and echinacoside at concentrations higher than the detection limits (0.05 mg g⁻¹ dry weight). Echinacoside, cynarin and chlorogenic acid were found in root tissues at concentrations ranging from 0.36 to 5.25 mg g⁻¹ dry weight. In all plant samples, echinacoside, which is the marker compound for E. angustifolia material, did not reach the minimum quality standard (10 mg g⁻¹ dry weight) for the production of standardized extract. We concluded that short-cycle, high-density greenhouse hydroponic culture stimulates plant growth and root production in E. angustifolia, but it does not ensure sufficient CADs accumulation in dried roots.     

Differential expression of defence-related proteins in Vigna unguiculata (L. Walp.) seedlings after infection with Fusarium oxysporum

This work aimed to study the induction of defence proteins in cowpea seedlings during the first days after infection with the fungi Fusarium oxysporum f. sp. cubenses and F. oxysporum f. sp. phaseoli. Cowpea seeds, after disinfection, were transferred to Petri dishes containing 0.5% agar and, after germination, were infected with a drop of a suspension containing 0.5 × 10⁴ spores ml⁻¹. Seedlings were collected at 24, 48, 72 and 96 h after infection and were dissected into leaves, hypocotyls, roots, cotyledons and teguments, which were measured and weighed for morphometric analysis. The agar medium was also analyzed. Enzymatic assays of proteic extracts yielded antimicrobial peptides detected by Western blotting. The cowpea seedlings showed a complex pattern of induction and repression of defence proteins in response to infection by both pathogens. Furthermore, morphometric analysis showed differences between infected and control seedlings. Infected samples did not at any time exhibit chitinase activity, but did exhibit different β-1,3-glucanase and peroxidase activities. Western blotting for lipid transfer protein (LTP) demonstrated its presence in all parts of the infected seedlings. Exuded proteins, also obtained from cowpea seeds in the germination medium, were separated by SDS-PAGE and tricine gel electrophoresis. The analysis showed that some proteins were exuded from moistened cowpea seeds, particularly after F. oxysporum infection.