María Rosa Simón

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.

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 .

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.

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.

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.

Mapping quantitative resistance to septoria tritici blotch in spelt wheat

The foliar wheat disease septoria tritici blotch can cause significant yield losses. A source of resistance has been mapped on chromosome 7D of spelt wheat, Triticum aestivum L. subsp. spelta (L.) Thell. The microsatellite-based genetic map was constructed from a set of 87 single-chromosome recombinant doubled-haploid lines bred from the cross between the landrace ‘Chinese Spring’ and a ‘Chinese Spring’-based line carrying chromosome 7D from spelt wheat. Two regions of the chromosome were associated with isolate-specific QTL expressed one at the seedling and another at the adult plant stage. The seedling resistance locus QStb.ipk-7D1 was found in the centromeric region of chromosome 7D, which corresponds to the location of the major resistance genes Stb4 originating from bread wheat cultivar ‘Tadinia’ and Stb5 originating from Triticum tauschii. The adult resistance locus QStb.ipk-7D2 was found on the short arm of chromosome 7D in a similar position to the locus Lr34/Yr18 known to be effective against multiple pathogens. Composite interval mapping confirmed QStb.ipk-7D1 and QStb.ipk-7D2 to be two distinct loci.

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.