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 P-1,3-glucanase and P-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 secreción 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 analizó luego de cultivarlas en medio líquido usando paredes celulares de R. solani como única fuente de carbono. Los resultados mostraron que Th11A80.1 y Th11C40.1 aumentaron la secreción de endoproteasas 2,77 y 4,79 veces, respectivamente; Th12A10.1 aumentó 1,86 veces la secreción de ß-1,3-glucanasa y 1,54 veces la de ß-1,4-quitinasa al compararlas con las cepas parentales. Se discute el rol de estas tres enzimas para el biocontrol de fitopatógenos.

J.R. Montealegre, F. Ochoa, X. Besoain, R. Herrera, and L.M. Pérez. 2014. In vitro and glasshouse biocontrol of Rhizoctonia solani with improved strains of Trichoderma spp. Cien. Inv. Agr. 41(2):197-206. 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.

J.R. Montealegre, F. Ochoa, X. Besoain, R. Herrera y L.M. Pérez. 2014. Biocontrol de Rhizoctonia solani in vivo y en invernadero, con cepas mejoradas de Trichoderma spp. Cien. Inv. Agr. 41(2):197-206. Se comparó la capacidad de cepas de Trichoderma spp., obtenidas previamente por fusión de protoplastos, con la de sus correspondientes cepas parentales, para biocontrolar a Rhizoctonia solani. El efecto biocontrolador se analizó sobre dos cepas de R. solani: 509 (GA 2-1) y 618 (GA 4). Las cepas producto de fusión de protoplastos de Trichoderma spp. fueron más efectivas que al menos una de las correspondientes cepas parentales. La inhibición más alta en experimentos de cultivos duales, se observó 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, sólo logró un 60,19% de inhibición con la cepa ThF2-1. El efecto inhibidor del desarrollo se debió principalmente a la secreción de metabolitos difusibles. El porcentaje de mortalidad y nivel de cancro en plantas de tomate se evaluó en experimentos de invernadero, en los que todas las cepas producto de la fusión 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.

Biorend SC (quitosana), BC-1000 EC (extrato de toranja mais flavonóides), e ECO-100 SC (bioflavonóides mais ácidos orgânicos, fitoalexinas cítricas, ácidos graxos glicerídeos e açúcares) inibiram em 80,1%, 79,0% e 76,5%, respectivamente, a podridão causada por Botrytis cinerea quando utilizados no tratamento pós-colheita de frutos de maçã sob condições controladas.Tratamento combinado de Biorend SC, com aplicação tanto em pré como no pós-colheita, proporcionou 49,9% de inibição da podridão, enquanto BC-1000 e ECO-100 EC não foram efetivos. Nenhum desses produtos inibiu a podridão cinzenta, quando utilizados em tratamento de pré-colheita em condições controladas ou em tratamento de pós-colheita em condições comerciais. O polissacarídeo algal ulvana, utilizado nos tratamentos de pós-colheita, reduziu em 56% a podridão cinzenta das maçãs em condições controladas, mas não teve efeito inibitório nos tratamentos combinados de pré e pós-colheita. A incapacidade dos produtos em ativar mecanismos de defesa (quitinases e peroxidases) nos frutos, após o tratamento em pré-colheita, foi consistente com a falta de controle da podridão nesse tipo de ensaio. Pelos resultados, sugere-se que os produtos naturais utilizados apresentam potencial para a utilização no manejo integrado da podridão de Botrytis quando aplicados em pós-colheita.

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.

Wild (Th11, Th12 and Th650) and mutant (Th11A80.1, Th12A40.1, Th12C40.1 and Th650-NG7) Trichoderma harzianum strains were stored for 180 days at 5ºC or at 22ºC, in two types of soils. Strains recovered at 90, 120 and 180 days from the two types of soils, retained their full capacity to biocontrol Rhizoctonia solani 618, that produces crown and root rot of tomatoes. Recovery, estimated as colony forming units (cfu) of the wild and mutant strains, showed that all increased their cfu after storage independently of the type of soil and temperature, although kinetic behavior differed among strains. Ratios of recovery after storage in type B soil/ type A soil or at 22ºC/5ºC, higher or lower than one respectively, allowed to establish that Th11 and Th12 were the most appropriate strains for the biocontrol of R. solani in conditions where growth of the phytopathogen is optimal.

IP3 increase and de novo synthesis of scoparone are produced in the hypersensitive response (HR) of lemon seedlings against the fungus Alternaria alternata. To elucidate whether a G-protein and/or a protein tyrosine kinase (PTK) are involved in signal transduction leading to the production of such a defensive response, we studied the HR in this plant system after treatment with G-protein activators alone and PTK inhibitors in the presence of fungal conidia. No changes in the level of IP3 were detected in response to the treatment with the G-protein activators cholera toxin or mastoparan, although the HR was observed in response to these compounds as determined by the scoparone synthesis. On the contrary, the PTK inhibitors lavendustin A and 2,5-dihidroxy methyl cinnamate (DHMC) not only prevented the IP3 changes observed in response to the fungal inoculation of lemon seedlings but also blocked the development of the HR. These results suggest that the IP3 changes observed in response to A. alternata require a PTK activity and are the result of a G-protein independent Phospholipase C activity, even though the activation of a G-protein can also lead to the development of a HR. Therefore, it appears that more than one signaling pathway may be activated for the development of HR in lemon seedlings: one involving a G-protein and the other involving a PTK-dependent PLC.

In addition to phytoalexin synthesis, the defense response of intact Citrus limon seedlings against Alternaria alternata involves both constitutive and induced enzyme activities such as chitinases (Ch) and ß-1,3-glucanases (Glu). A. alternata conidial germination was prevented by protein extracts from inoculated lemon seedlings, but also by extracts from mock-inoculated specimens. On the other hand, degradation of mycelia was accomplished only by protein extracts from inoculated seedlings. The presence of six Ch isoenzymes and of four Glu isoenzymes was detected in protein extracts from mock-inoculated seedlings. As a result of fungal inoculation, the isoenzyme pattern of Ch and Glu changed, making possible the detection of a new Ch isoenzyme and of three new Glu. Also, some constitutive Ch and Glu increased their enzyme activity, and those Ch that increased their activy also showed a broadening of their substrate specificity. These changes were prevented by a-amanitin and cycloheximide, suggesting that the presence of new Ch and Glu isoenzymes was due to de novo synthesis of proteins. Results suggest that constitutive Ch and Glu could act as pre-formed defense molecules in Citrus limon preventing A. alternata germination, while those induced after fungal inoculation of lemon seedlings could act along with the former, to produce lysis of fungal mycelia, resulting in a more efficient control of A. alternata development..

Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3 ) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. 3.1.4.3) inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata