Analía Perelló

DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties

Species of the anamorph genus Phoma are commonly isolated from a wide range of ecological niches. They are notoriously difficult to identify due to the paucity of morphological features and the plasticity of these when cultivated on agar media. Species linked to Phoma section Peyronellaea are typified by the production of dictyochlamydospores and thus have additional characters to use in taxon delineation. However, the taxonomy of this section is still not fully understood. Furthermore the production of such chlamydospores also is known in some other sections of Phoma. DNA sequences were generated from three loci, namely ITS, actin, and β-tubulin, to clarify the phylogeny of Phoma taxa that produce dictyochlamydospores. Results were unable to support section Peyronellaea as a taxonomic entity. Dictyochlamydospore formation appears to be a feature that developed, or was lost, many times during the evolution of Phoma. Furthermore, based on the multigene analyses, five new Phoma species could be delineated while a further five required taxonomic revision to be consistent with the genetic variation observed.

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.

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 .

Pathogenic and molecular variability among isolates of Pyrenophora tritici-repentis , causal agent of tan spot of wheat in Argentina

Tan spot, caused by Pyrenophora tritici-repentis, is a common disease of wheat (Triticum aestivum) responsible for economic losses in some wheat growing areas worldwide. In this study the pathogenic and genetic diversity of 51 P. tritici-repentis isolates collected from different ecological regions of Argentina were analyzed. Virulence tests were conducted on 10 selected wheat cultivars: Buck Halcón, Chris, Gabo, Glenlea, Klein Dragón, Klein Sendero, Max, ND 495, ProInta Guazú and ProInta Imperial. Data revealed significant differences between all main factors evaluated and the interactions for 19 of the isolates analyzed. Based on the reaction type of each isolate/cultivar combination, 48 different pathogenic patterns were detected. The molecular analysis using Inter-Simple Sequence Repeats (ISSR) revealed the existence of 36 different haplotypes among 37 isolates of P. tritici-repentis originally selected for this study. These results indicate that P. tritici-repentis on wheat in Argentina is a heterogeneous fungus, implying that screening wheat germoplasm for resistance for tan spot disease requires a wide range of pathogen isolates.

Tan spot of wheat (Triticum aestivum L.) infection at different stages of crop development and inoculum type

Abstract The incidence, severity and progress of tan spot ( Pyrenophora tritici-repentis ) of wheat under different inoculum types were analysed during 2000 and 2001 in two different environments of Buenos Aires Province. Severity, incidence and area under the disease progress curve (AUDPC) were higher in 2001 than in 2000. Inoculum as spore pulverization treatments of Drechslera tritici-repentis occasioned higher disease severity than the application of oat grains colonized with the pathogen at low or high inoculum concentration. Severities increased with the growth stage of wheat plants. In both environments severity was higher when the evaluation was performed at 14 days compared to 7 days after inoculation. The combined analysis for incidence and severity showed significant differences between all main factors and the interactions except the quadruple for incidence and environment×evaluation date and environment×growth stage×evaluation date for severity. For the AUDPC all main factors and interactions were significant except environment×evaluation date. Due to the significant interactions with the environment both years were analysed separately. For the separate analysis, incidence, severity and AUDPC increased with the growth stage and evaluation date in both years. The severity and AUDPC increased for the spore pulverization treatment with respect to oat grain application. For incidence there were differences between both treatments only in 2000 environment. Furthermore, the highest rate of spore pulverization caused a higher severity and AUDPC than the lowest rate. However, there were no differences for incidence between doses.

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.

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.

Greenhouse Screening of the Saprophytic Resident Microflora for Control of Leaf Spots of Wheat (Triticum aestivum)

Ten microorganisms of the epiphytic microflora of wheat leaves in Buenos Aires Province, Argentina, were evaluated under greenhouse conditions as potential biocontrol agents of the pathogensAlternaria triticimaculans, Bipolaris sorokiniana, Drechslera tritici-repentis andSeptoria tritici in two application sequences (prior to or together with the pathogens). The antagonists significantly reduced the expression of the diseases on wheat plants compared with control plants not inoculated with the antagonists. Maximum percentage of reduction of the necrotic lesion area (NLA) (40–55%) ofS. tritici resulted whenCryptococcus sp.,Rhodotorula rubra andPenicillium lilacinwn were sprayed on leaves prior to inoculations with the pathogen.Bacillus sp.,Cryptococcus sp.,Fusarium moniliforme var.anthophylium,P. lilacinum andR. rubra reduced significantly (34–52%) the NLA ofB. sorokiniana in both of the application sequences. The best antagonistic effect againstA. triticimaculans was shown byAspergillus niger, Bacillus sp.,Chaetomium globosum, F. moniliforme var.anthophylium andNigrospora sphaerica, with a NLA reduction from 21% to 35% in the co-inoculation or in the sequential application. All microorganisms exceptN. sphaerica performed better than the control againstD. tritici-repentis. The area under disease progress curve (AUDPC) of the pathogens appeared to progress similarly, but at lower values, in treated plants than in untreated controls. The two yeasts and the bacteria decreased AUDPC to 50–55% ofS. tritici andB. sorokiniana compared with the control in both application sequences, whereas the maximum efficacy againstA. triticimaculans was reached byN. sphaerica andA. niger for the sequential application and byF. moniliforme var.anthophylium for the co-inoculation. If the parasitism occurs also in nature, application of antagonists for biological control might provide the opportunity to compete with the pathogens and regulate their colonization in wheat leaves.