Jaime Montealegre

Phytoplasmas Associated with Grapevine Yellows Disease in Chile

An extensive survey was performed from 2002 to 2006 to detect and identify phytoplasmas associated with Chilean grapevines. Nested polymerase chain reaction assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R2 detected phytoplasmas in 34 out of the 94 samples tested (36%). Restriction fragment length polymorphism (RFLP) analyses, cloning, and sequencing allowed identification of phytoplasmas belonging to ribosomal subgroups 16SrI-B, 16SrI-C, 16SrVII-A, and 16SrXII-A. The 16SrVII-A phytoplasma represents a new finding in grapevine; moreover, variability of the RFLP profile was observed in some of the 16SrXII-A phytoplasmas, indicating possible new ribosomal subgroups. Mixed phytoplasma infections and infections of phytoplasmas together with one or more viruses also occurred.

Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus

Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.

The expression of extracellular fungal cell wall hydrolytic enzymes in different Trichoderma harzianum isolates correlates with their ability to control Pyrenochaeta lycopersici

Four isolates of Trichoderma harzianum (ThN3, Th11, Th12 and Th16) were selected for their ability to control the in vitro development of the tomato root pathogen Pyrenochaeta lycopersici. Analysis of the mechanisms involved in biocontrol showed that the formation of non-volatile metabolites appears to be one of those involved in biocontrol of P. lycopersici by all T. harzianum isolates tested. Nevertheless, the higher secretion of chitinases, both in number of isoenzymes and activity by the Th11 strain, correlated well with its higher ability to control this agent in laboratory and greenhouse experiments as compared to the other T. harzianum isolates tested. The secretion of beta-1,3-endoglucanases and/or proteases appeared to have less significance than endochitinases in the biological control of P. lycopersici.

Control of grey rot of apple fruits by biologically active natural products

Biorend SC (chitosan), BC-1000 EC (grapefruit extract plus bioflavonoids) and ECO-100 SC (bioflavonoids plus organic acids, citric phytoalexins, fatty acids, glycerides and sugars), respectively, suppressed grey rot of apple caused by B. cinerea by 80.1%, 79.0% and 76.5% when used as post-harvest treatments under controlled conditions. When applied as combined pre- and post-harvest treatments Biorend SC inhibited fruit rot by 49.9 %, while BC-1000 EC and ECO-100 SC were ineffective. None of the products inhibited fruit rot when applied as pre-harvest treatments under controlled conditions or as post-harvest treatments under commercial conditions. The algal polysaccharide ulvan used in post-harvest treatments suppressed grey rot by 56.0% under controlled conditions, but had no inhibitory effect on combined pre- and post-harvest treatments. The inability of products to activate defense mechanisms (chitinase and peroxidase) of fruits was consistent with the unsuccessful control of rot by pre-harvest treatment. The results suggest that the natural products used have potential for use in integrated management of Botrytis rot when applied after harvest.

New strains obtained after UV treatment and protoplast fusion of native Trichoderma harzianum: their biocontrol activity on Pyrenochaeta lycopersici

The obtainment of 30 new strains from native Trichoderma harzianum after UV light irradiation (UV-A and UV-C), and of 82 strains resulted from protoplast fusion were accomplished. The new strains, initially selected for their growing rate under low temperature and high pH conditions, as well as for their innocuousness on tomato plants, were tested for in vitro inhibition of Pyrenochaeta lycopersici in dual cultures and due to secretion of volatile and diffusible metabolites. All the UV-A and UV-C selected candidate mutants were innocuous to tomato plants, but none of them showed improvement in their biocontrol activity on P. lycopersici . Th12A20.1 increased 1.3 and 1.9 fold the total fresh weight of Fortaleza tomato plants when compared to its parental strains Th12 and Th11, respectively. The selected candidate mutants obtained through protoplast fusion were also innocuous to tomato plants, but only ThF1-2 and ThF4-4 inhibited 1.3 fold (in dual cultures) and 5 fold (due to secretion of volatile metabolites) the growth of P. lycopersici , respectively, in relation to the mean inhibitory effect of both parents. Therefore, these candidate mutants could be included in experiments under field conditions.

Biocontrol capacity of wild and mutant Trichoderma harzianum (Rifai) strains on Rhizoctonia solani 618: effect of temperature and soil type during storage

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.12 No.4, Issue of October 15, 2009

Sensitivity of wild-type and mutant Trichoderma harzianum strains tofungicides

R. Herrera, D. Nunez, N. Romero, X. Besoain, L. M. Perez, and J. Montealegre. 2012. Sensitivity of wild-type and mutant Trichoderma harzianum strains to fungicides. Cien. Inv. Agr. 39(3): 569-576. The germination of conidia in wild-type (Th11, Th12 and Th650) and mutant (Th11A80.1, Th12A10.1 and Th650-NG7) strains of Trichoderma harzianum that were exposed to different commercial fungicides was studied. All wild-type and mutant Trichoderma strains were germinated in the presence of 1,700 mg L -1 of pencycuron. The wild-type strains Th12 and Th650 and the corresponding mutant strains Th12A10.1 and Th650-NG7 were sensitive to all concentrations of iprodione and metalaxil + mancozeb. The EC 50 (Median Effective Concentration) values for the different fungicides were between 10 -1 and 10 -4 less than the concentrations recommended for field applications; one exception was Phyto ‑fos on Th650‑NG7, where this ratio was 0.72. These results establish whether some of these fungicides can be used in combination with the biocontrol agents evaluated.

Enhanced secretion of biocontrol enzymes by Trichoderma harzianum mutant strains in the presence of Rhizoctonia solani cell walls

The secretion of enzymes involved in biocontrol from the wild T. harzianum strains Th11 and Th12 and the mutants Th11A80.1, Th11C40.1 and Th12A10.1 was studied after their cultivation in the presence of R. solani cell walls as the sole carbon source. The results showed that endoprotease activity in the supernatants from Th11A80.1 and Th11C40.1 increased 2.77 and 4.79-fold, respectively, and the ‑1,3‑glucanase and -1,4-chitinase activity from Th12A10.1 increased 1.86 and 1.54-fold, respectively, compared with the corresponding parental strains. The role of the three enzymes in the biocontrol of R. solani is discussed. La secrecion de enzimas involucradas en biocontrol por las cepas silvestres de T. harzianum Th11 y Th12, y de sus mutantes Th11A80.1, Th11C40.1 y Th12A10.1, se analizo luego de cultivarlas en medio liquido usando paredes celulares de R. solani como unica fuente de carbono. Los resultados mostraron que Th11A80.1 y Th11C40.1 aumentaron la secrecion de endoproteasas 2,77 y 4,79 veces, respectivamente; Th12A10.1 aumento 1,86 veces la secrecion de ‑1,3‑glucanasa y 1,54 veces la de ‑1,4‑quitinasa al compararlas con las cepas parentales. Se discute el rol de estas tres enzimas para el biocontrol de fitopatogenos.

In vitro and glasshouse biocontrol of Rhizoctonia solani with improved strains of Trichoderma spp.

The potential of Trichoderma spp. fusants for the biocontrol of Rhizoctonia solani was compared with the ability of their corresponding parental strains. Their effect was tested in vitro using two R. solani strains, 509 (AG 2-1) and 618 (AG 4). The highest inhibitions in growth in dual cultures were obtained with the ThF2-1 (89.79%), ThF3-3 (90.55%), ThF4-15 (91.75%) and ThF5-8 (77.67%) fusants on R. solani 509; only ThF2-1 was able to inhibit the growth of R. solani 618 (60.19%). The inhibitory effect on growth was mainly due to diffusible metabolites. Percent mortality and canker level in tomato plants were evaluated in glasshouse experiments where all of the evaluated fusants suppressed plant mortality, but only ThF2-1 and ThF5-8 significantly decreased the canker level. Se comparo la capacidad de cepas de Trichoderma spp., obtenidas previamente por fusion de protoplastos, con la de sus correspondientes cepas parentales, para biocontrolar a Rhizoctonia solani . El efecto biocontrolador se analizo sobre dos cepas de R. solani : 509 (AG 2-1) y 618 (AG 4). Las cepas producto de fusion de protoplastos de Trichoderma spp. fueron mas efectivas que al menos una de las correspondientes cepas parentales. La inhibicion mas alta en experimentos de cultivos duales, se observo con las cepas ThF2-1 (89,79%), ThF3-3 (90,55%), ThF4-15 (91,75%) y ThF5-8 (77,67%) sobre R. solani 509; mientras que el efecto biocontrolador sobre R. solani 618, solo logro un 60,19% de inhibicion con la cepa ThF2-1. El efecto inhibidor del desarrollo se debio principalmente a la secrecion de metabolitos difusibles. El porcentaje de mortalidad y nivel de cancro en plantas de tomate se evaluo en experimentos de invernadero, en los que todas las cepas producto de la fusion de protoplastos de Trichoderma spp. suprimieron la mortalidad de plantas de tomate, y solamente ThF2-1 y ThF5-8 disminuyeron significativamente el nivel de cancro.