Cristina Alicia Cordo

Molecular mapping of quantitative trait loci determining resistance to septoria tritici blotch caused by Mycosphaerella graminicola in wheat

A set of 65 recombinant inbred lines of the ‘International Triticeae Mapping Initiative’ mapping population (‘W7984’בOpata 85’) was analysed for resistance to septoria tritici blotch at the seedling and adult plant stages. The mapping population was inoculated with two Argentinean isolates (IPO 92067 and IPO 93014). At the seedling stage, three loci were discovered on the short arms of chromosomes 1D, 2D and 6B. All three loci were detected with both isolates. At the adult plant stage, two isolate-specific QTL were found. The loci specific for isolates IPO 92067 and IPO 93014 were mapped on the long arms of chromosomes 3D and 7B, respectively.

Mating-type Distribution and Fertility Status in Magnaporthe grisea Populations from Argentina

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M.grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.

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.

Chromosomal location of resistance to Septoria tritici in seedlings of a synthetic hexaploid wheat, Triticum spelta and two cultivars of Triticum aestivum

Chromosomal location of resistance to two virulent Argentinean isolatesof Septoria tritici was studied in two wheat (Triticum aestivumL.) cultivars (Cappelle-Desprez & Cheyenne), a synthetic hexaploid(Synthetic 6x) and Triticum spelta in seedlings. Substitution lines of these(resistant or moderately resistant) genotypes into (susceptible) ChineseSpring were selected from a previous screening. For Synthetic 6x,resistance was clearly located in chromosome 7D. Chinese Spring with the7D chromosome substituted by Synthetic 6x showed almost completeresistance, similar to the level of Synthetic 6x. For the substitutions withCappelle-Desprez, Cheyenne, and T.spelta there were no lines with abehaviour similar to the resistant parent. However, some substitutions weremore resistant than the susceptible parent suggesting that severalchromosomes could be involved in the resistance of these genotypes toSeptoria leaf blotch.

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.

A conventional PCR technique to detect Septoria tritici in wheat seeds

A PCR assay was developed for detection of wheat seed naturally contaminated with Septoria tritici. S. tritici specific primers were derived from strict alignment of ITS and α-tubulin sequences of the pathogen. The specificity of four sets of synthesised oligonucleotide pairs (A, B, C and D) were tested using isolates from S. tritici, other selected fungi and wheat seeds. A single DNA fragment was amplified from S. tritici isolates with all primer pairs, whereas no product was generated from other DNA sources. S. tritici was also detected in wheat seed lots collected from plants with variable pycnidial coverage on the upper two leaves. PCR detection of as little as 0.5 pg of S. tritici genomic DNAwas possible. This is the first report on the detection of S. tritici DNA in naturally infested wheat seeds. This PCR based assay is simple, rapid, specific, sensitive and suitable for routine detection of the wheat pathogen in infested wheat seeds.

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.

DNA fingerprint and pathotype diversity of Pyricularia oryzae populations from Argentina

The genetic diversity of the rice blast pathogen, Pyricularia oryzae, was analysed in rice-growing provinces of Argentina. A total of 161 isolates of the fungus was collected from 15 rice cultivars at nine locations during 2000-05 and characterised using Pot2-DNA fingerprinting. Based on DNA analysis (isolates with ≥70% band similarity), five lineages were identified and designated A, B, C,Dand E, with 11, 22, 4, 1 and 4 haplotypes identified, respectively. The predominant lineage, B, representing 38% of the collected isolates, was recovered from four cultivars in five locations. In contrast to lineages A and B, which did not contain a dominant haplotype, a single haplotype predominated in lineages C and E. Isolates representing all haplotypes were examined for virulence on a set of differential rice cultivars, near-isogenic lines and commercial cultivars commonly grown in Argentina, revealing 41 pathotypes and 24 international races. There was no significant association between DNA fingerprint similarities and pathotypes. Overall, these data indicated that populations of P. oryzae in Argentina are genetically simple and predominantly clonal yet have a high pathotype diversity.