Cecilia Inés Mónaco

Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms

Fusarium graminearum is associated with the cereal damping-off complex which reduces germination, seedling stand and yield. Fifty-two bacterial strains and six Trichoderma spp. isolated from the wheat rhizosphere were evaluated for biocontrol of seedling blight of wheat caused by F. graminearum. Their potential as biocontrol agents was tested in vitro and in the greenhouse. Isolates varied in their ability to inhibit the mycelial growth of F. graminearum in agar plate bioassays by 0–79%. This parameter was not related with biocontrol efficacy of in vivo assays. In greenhouse trials, all isolates were initially evaluated for reducing disease on wheat cultivars Klein Centauro (moderately resistant to F. graminearum) and Pro INTA Oasis (susceptible) planted in sterilized soil artificially infested with the pathogen. Among the 25 bacteria and six fungal isolates that exhibited a pronounced suppressive effect, the most efficient 10 for both cultivars were further assayed on eight cultivars (Buck Candil, Buck Catriel, Buck Chambergo, Buck Poncho, Buck Topacio, Klein Cacique, Klein Centauro and Pro INTA Oasis) potted in cultivated–inoculated soil. Three weeks after sowing, plant stand, percentage of diseased emerging seedlings, plant height and dry weight were evaluated. Among the antagonists only Stenotrophomonas maltophilia was significantly better than the control for the average of the eight cultivars for plant stand, height and dry weight. Stenotrophomonas maltophilia also caused a non-significant decrease in the percentage of diseased plants. Three strains of Bacillus cereus and one isolate of Trichoderma harzianum gave also a good control in some cultivars. The ability of these isolates to affect the infection of wheat seedlings by F. graminearum may be of potential value in field trials.

Biocontrol efficacy of Trichoderma isolates for tan spot of wheat in Argentina

Abstract Pyrenophora tritici-repentis (Died) Drechs. (anamorph= Drechslera tritici-repentis (Died) Shoem. is one of the most important and widespread necrotrophic pathogen of wheat in Argentina. Breeding, chemical treatments and appropriate cultural practices are the main ways for disease control. The possibility of biological control is added as a complementary strategy within the integrated management of the disease. Trichoderma spp. have been used as biocontrol agents to protect plants against soil-borne and foliar diseases in several crops. The objective of the present work was to evaluate the potential of Trichoderma harzianum (isolate Th15, Th11, Th2, Th81, Th7, Th13, Th8, Th5), Trichoderma aureoviride (isolate Ta1, Ta100) and Trichoderma koningii (isolate Tk11, Tk6) as biocontrol agents of D. tritici-repentis under in vitro and greenhouse conditions. Dual cultures in Petri dishes containing potato dextrose agar showed that the isolates of Trichoderma spp. tested inhibited significantly the mycelial growth of D. tritici-repentis between 50% and 74%. Microscopic examination of cultures of Trichoderma spp. and D. tritici-repentis in close proximity showed plasmolysis of conidia and hyphae of the pathogen. The results of the greenhouse tests in 2000 and 2001 indicated that seven strains of Trichoderma spp. (Th5, Th11, Th13, Tk6, Tk1, Th2 and Th81) significantly reduced the disease severity on wheat plants compared with untreated plants. In general, there was a significant decrease in Trichoderma spp. population on the wheat phylloplane with time. Additional greenhouse studies using other isolates and under a wide range of temperature conditions are needed to fully assess the potential and limitations of Trichoderma spp. as biocontrol agents of D. tritici-repentis .

Preliminary studies on biological control of the blackpoint complex of wheat in Argentina

Blackpoint is a brownish or black discoloration of wheat kernels and biological control is a complementary strategy to manage the disease. This work evaluated the effect of five strains of Trichoderma harzianum and one strain of T. koningii on the growth of Bipolaris sorokiniana and Alternaria alternata and compared the results of screening tests under controlled conditions and field evaluations on bread and durum wheat ears. Disease incidence, infection percentage and seedling emergence percentage determined in a greenhouse assay were evaluated. Dual cultures showed Trichoderma spp. inhibited significantly the mycelial growth of B. sorokiniana between 36 and 71% and of A. alternata between 41 and 61%. Microscopic examination of B. sorokiniana and A. alternata showed plasmolysis and vacuolization of hyphae of the pathogens in the presence of the antagonists tested. With pre-inoculation of wheat ears at anthesis under field conditions, disease incidence, infection percentage by blotter tests and seedling emergence in the greenhouse did not show significant differences between controls and treatments with Trichoderma spp.

Trichoderma spp. as elicitors of wheat plant defense responses against Septoria tritici

Abstract Leaf blotch of wheat is a widespread and highly active disease that affects wheat production. In addition to the use of chemicals and proper cultivation methods, microbial antagonists are used to control plant pathogens. Trichoderma spp. stimulate a systemic induced response in plants. Therefore, the efficacy of Trichoderma spp. against wheat leaf blotch was evaluated under greenhouse conditions. The susceptible plants were sprayed with Septoria tritici conidiospores. In order to select an efficient method of pretreatment with Trichoderma spp., leaf spraying and seed coating with 14 isolates were tested in 2003 and 2004. The extent of leaf necrosis area and pycnidial coverage was estimated. Antagonism was assessed by the capacity of each Trichoderma spp. isolate to restrict the progress of leaf blotch, 21 days after inoculation. Of the two methods, seed coating was more efficacious against leaf blotch than leaf spraying. Amongst the 14 isolates tested, the isolate prepared from T. harzianum (Th5) produced the highest level of protection. None of the treatments caused changes in plant stem diameter or dry weight. Trichoderma spp. did not get into leaves while S. tritici was present, even in asymptomatic leaf extracts. In addition, the leaf apoplast antifungal proteolytic activity was measured in plants 7, 15, and 22 days after sowing. This antifungal action decreased in plants only inoculated with S. tritici, but increased in those grown from seeds coated with the T. harzianum (Th5) isolate. This increase conferred resistance to the susceptible wheat cultivar. The endogenous germin-like protease inhibitor coordinated the proteolytic action. These results suggest that T. harzianum stimulates a biochemical systemic induced response against leaf blotch.

Biological control of Septoria tritici blotch on wheat by Trichoderma spp. under field conditions in Argentina

Biological control is an additional tool available for the design of more sustainable control strategies of wheat diseases. Trichoderma spp. have previously been used as biocontrol agents to protect wheat plants against leaf spots diseases in Argentina, but the information from field assays is scarce. The effectiveness of four Trichoderma harzianum strains and one T. koningii strain in reducing the incidence and severity of the leaf blotching of wheat caused by Septoria tritici blotch (STB) under two formulation conditions, spore suspension and the coated-seed technique, was studied under field conditions. Significant differences between wheat cultivars, formulation types and growth stages were found. In 2003, at the tillering stage, all of the treatments tested (except SST1 for incidence) effectively reduced the incidence or the severity of the disease compared to the control. Similarly, in 2004, ten of the treatments reduced the severity at tillering. At the heading stage, none of the treatments tested caused a significant decrease of the disease. These results indicated, therefore, that the antagonism was effective at an early stage of the disease only. Comparing both formulations, spraying spore suspension onto leaves and the coated-seed application technique, both were effective in decreasing the disease. Some isolates, such as CST4 and CST2, reduced the incidence value of STB to 40% and the severity value to 70% of the control values applied as coated-seed formulation. On the other hand, isolates T4 and T2 showed the greatest effectiveness for controlling STB, with similar reduction values to that shown by the fungicide (Folicur®) application treatment. The results of this study indicated that, although the immediate impact of Trichoderma isolates may be seen as reduced incidence and severity on the first stages of STB, in the long term, the same disease levels as found in untreated sites may be attained. This study also demonstrated that the incorporation of Trichoderma as a biocontrol preparation may be a promising step towards reducing STB disease in the field and the levels of fungicide residues in the context of a more integrated approach to the problem.

Characterization of novel Trichoderma spp. isolates as a search for effective biocontrollers of fungal diseases of economically important crops in Argentina

Monoconidial cultures of 33 isolates of Trichoderma from Buenos Aires Province, Argentina were characterized on the basis of twenty eight morphological, physiological and biochemical features. All of them were screened for proteinase, endochitinase and β-1,3 glucanase activity. Universally primed PCR (UP-PCR) and inter-simple sequence repeat (ISSR) techniques were used to examine the genetic variability among isolates, which resulted in 127 bands for the total number of isolates. These results were subjected to numerical analysis revealing 20 haplotypes grouped in five clusters. The ability of Trichoderma isolates to antogonize soil-borne fungal plant pathogens using a dual culture assay was done against five fungal species: Alternaria sp., Bipolaris sorokiniana, Fusarium graminearum, F. solani, and Pyricularia oryzae. The highest inhibition values (85% RI) were obtained against B. sorokiniana and P. oryzae. Three isolates of T. harzianum named as FCCT2, FCCT3 and FCCT9 were capable of causing a high growth inhibition on four of the fungal species assayed, which was in agreement with their higher extracellular hydrolytic activity. Our results suggest that these isolates have the potential to be effective agents for biocontrol of cereal and tomato fungal pathogens.

The effect of Trichoderma harzianum and T. koningii on the control of tan spot (Pyrenophora tritici-repentis) and leaf blotch (Mycosphaerella graminicola) of wheat under field conditions in Argentina

Abstract The effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot (Pyrenophora tritici-repentis) and leaf blotch of wheat (Mycosphaerella graminicola) was evaluated under field conditions. Significant differences between wheat cultivars, inoculum types and growth stages were found. Three of the isolates tested (T2 for M. graminicola, T7 for P. tritici-repentis and T5 for both of them) showed the best performance in controlling leaf blotch and tan spot when coated onto seed or sprayed onto wheat leaves at different growth stages, with significant severity reduction up to 56%. At tillering, six of the isolates reduced the severity of P. tritici-repentis and M. graminicola compared to the control by up to 39% and 12–53%, respectively. In some experiments, the biocontrol preparation (T2 and T5) gave a level of disease control similar to that obtained with Tebuconazole (70 and 48%, respectively). The effect of Trichoderma against P. tritici-repentis was also observed at the heading stage, when six of the treatments reduced disease severity by 16–35%. This is the first report on the efficacy of Trichoderma spp. against wheat necrotrophic pathogens under field conditions in Argentina.

Antagonistic effect of soil rhizosphere microorganisms on Bipolaris sorokiniana, the causal agent of wheat seedling blight

The biocontrol efficiency of Epicoccum purpurascens, Gliocladium roseum, three strains of Bacillus subtilis, and Pseudomonas fluorescens, isolated from the rhizosphere of wheat plants, was assessed in relation to seedling blight caused by Bipolaris sorokiniana. An in vitro study of the potential antagonist was performed using the dual culture technique and by ‘sowing’ wheat seeds pelleted with the saprophytes in plates with water agar + the pathogen. In vivo assays were carried out in the greenhouse and in the field with pelleted seeds sown in artificially infested soil. Both the number of living plants and the number of plants with necrosis on the leaves and the base of the stems and roots were assessed 15 days after sowing. Under greenhouse conditions, B. subtilis 3 and G. roseum reduced the level of infection of Buck Pucará and Trigomax 100 cultivars, respectively. In the field, biocontrol of the disease was not achieved.

Effect of Trichoderma spp. isolates for biological control of tan spot of wheat caused by Pyrenophora tritici-repentis under field conditions in Argentina

Trichoderma spp. have been used as biocontrol agents to protect plants against foliar diseases in several crops, but information from field assays is scarce. In the present work, experiments were carried out to determine the effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot, caused by Pyrenophora tritici-repentis (anamorph: Drechslera tritici-repentis) under field conditions. Significant differences between years, wheat cultivars and treatments were found. In 2003, two of the isolates assayed (T5, T7) showed the best performance against the disease applied as seed treatments or sprayed onto wheat leaves at different stages. The application of six of the treatments on wheat plants significantly reduced disease severity by 16 to 35% in comparison with the control. Disease control provided by isolate T7 was similar to that provided by the fungicide treatment (56% reduction). This is the first report on the efficacy of Trichoderma spp. against tan spot under field conditions in Argentina.

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.