Biocontrol of root and crown rot in tomatoes under greenhouse conditions using Trichoderma harzianum and Paenibacillus lentimorbus. Additional effect of solarization Jaime
R. Montealegre Rodrigo
Herrera Juan
Carlos Velásquez Polyana
Silva Ximena
Besoaín Luz
María Pérez* *Corresponding author Financial support: Fondecyt 1990785. Keywords: Fusarium oxysporum, Lycopersicon esculentum, Pyrenochaeta lycopersici, Rhizoctonia solani, solarization.
Trichoderma harzianum 650 (Th650) and Paenebacillus lentimorbus 629 (Pl629) selected earlier for their ability to control Rhizoctonia solani, Fusarium solani and F. oxysporum in vitro, were applied alone or combined with solarization (summer assay) and/or with methyl bromide (MeBr) (summer and winter assays) to a soil with a high inoculum level, for the control of tomato root rot caused by the complex F. oxysporum f. sp. lycopersici - Pyrenochaeta lycopersici - Rhizoctonia solani. Evaluations were also performed independently for root damage caused by P. lycopersici, and also for R. solani in the summer assay. MeBr decreased tomato root damage caused by the complex from 88.7% to 21.2% and from 78.4% to 35.7% in the summer and in the winter assay, respectively. None of the bio-controllers could replace MeBr in the winter assay, but Th650 and Pl629 reduced root damage caused by this complex in the summer assay. Treatments with bio-controllers were improved by their combination with solarization in this season. Independent evaluations showed that the positive control of Th650 towards R. solani and the lack of effect on P. lycopersici correlates well with the endochitinase pattern expressed by Th650 in response to these phytopathogens. Root damage caused by R. solani can be controlled at a similar level as it does MeBr in summer assays, thus representing an alternative to the use of this chemical fungicide for the control of this phytopathogen.
Root
and crown rot of tomatoes can be observed after attack by several
phytopathogens, includingFusarium oxysporum f. sp. lycopersici
and Rhizoctonia solani. In addition, corky root is developed
after attack by Pyrenochaeta lycopersici (Campbell
and Shishkoff, 1990). The presence of all these three pathogens
is commonly found in the rhizosphere of tomato plants cultivated under
greenhouse conditions and during the same season, in V Region - The
natural control of several phytopathogens is based on the presence
of suppressive soils where several biocontrol microorganisms are detected,
such as those belonging to Trichoderma, Gliocadium,
Pseudomonas and Bacillus genera, among others (Weller
et al. 2002; Montealegre et al. 2003; Guo
et al. 2004; Huang et al. 2005). In Our group has been working in biocontrol of several pathogens that infect tomato roots, selecting both bacteria and fungi with biocontrol activity (Reyes et al. 2000; Lespinasse et al. 2001; Montealegre et al. 2003). We have described the biocontrol of R. solani and of F. solani by an isolate of P. lentimorbus (Montealegre et al. 2003), and the biocontrol of P. lycopersici (Pérez et al. 2002), and of R. solani (Santander et al. 2003) by isolates of T. harzianum. Also, the effect of solarization on F. oxysporum, R. solani and P. lycopersici has been tested (Montealegre et al. 1996; Santander et al. 2003).Nevertheless, it is unknown whether the combination of selected native fungi (T. harzianum) and bacteria (P. lentimorbus) and the use of solarization are able to replace MeBr in the control of tomato root pathogens. The present work reports the biocontrol activity of T. harzianum 650 and of P. lentimorbus 629 and solarization for the control of the complex. Selection of the biocontrol agents was done based on previous results on R. solani and F. solani (Escobar et al. 2004) and taking into account that each Trichoderma isolate behaves differentially when confronted to the same pathogen (Pérez et al. 2002). Microorganisms and culture conditions: Th650 and Pl629 The
fungus was isolated from tomato monoculture suppressive soils. It
was cultured on potato-dextrose-agar, PDA (DIFCO), for the obtention
of conidia (Lu et al. 2004). These were used as
inoculum for liquid cultures of the fungus for: a) its characterization
in terms of the secretion of biocontrol enzymes; b) for obtention
of the inoculum to be applied to tomato seeds and c) for formulations.
The innoquity of this Trichoderma isolate on tomato seedlings
was tested as follows: a) tomato seeds of variety 593 were covered
with a sodium alginate based formulation containing Th650 (Montealegre
and Larenas, 1997) and placed in speedlings containing a previously
sterilized mixture of perlite: vermiculite = 1:1 (w/w); and b) tomato
seedlings were transplanted to speedlings containing the mixture already
mentioned plus The
bacterium was stored in tubes containing B King medium at Conidia from Th650 (1 x 106 ) were used to inoculate 200 mL of liquid Mandels medium using cell walls of R. solani or F. oxysporum or P. lycopersici as the sole carbon source, as described (Pérez et al. 2002). Supernatants from these cultures were used to characterize the isoenzymic pattern of hydrolytic enzymes involved in biocontrol (endochitinases, β-1,3-glucanases and proteases), secreted by this fungal isolate in response to the presence of cell walls of the different phytopathogens (Pérez et al. 2002). Briefly, native PAGE at pH 4.4 separated proteins (50 µg per lane) from supernatants of culture media. Endochitinase activity was visualised after incubating the polyacrylamide gel with an auxiliary 2% (w:v) agarose gel containing glycol chitin, and further incubation with 0.01% (w:v) fluorescent brightener 28. β-1,3-glucanase activity was visualised after incubating the gel with 1% (w:v) laminarin and developing bands of activity with 0.15% (w:v) triphenyltetrazolium. Protease activity was visualised after incubating an haemoglobin containing gel with Coomassie blue. Treatments and evaluations: Treatments, solarization, evaluations of the assays, determination of the inoculum of phytopathogens found in soils Treatments
were done under commercial greenhouse conditions. They were done applying
Th650 and Pl629 alone or combined with solarization and/or with methyl
bromide in a soil previously selected on the basis of its high inoculum
level content of P. lycopersici, of R. solani and of
the complex F. oxysporum f. sp. lycopersici - P.
lycopersici - R. solani in Quillota, V Region of Chile.
Two assays were done during the seasons 2001 (winter assay) and 2001-2002
(summer assay) using commercial crops of the tomato variety The
biocontrol assay in summer included the following treatments: Control
(no soil treatment); MeBr (Methylbromide plus chloropicrine (98:2
in %) using a dosage of 75.5 g/m2); Th650 ( MeBr
was applied on June 28 - July 3, followed by aeration up to July Solarization
was performed between November 28, 2001 and January 10, 2002, which
corresponds to late Spring - beginning Summer in The evaluation of the assays (depending on the soil used for them) considered: a) Root damage (corky root) caused by P. lycopersici (Campbell and Shishkoff, 1990); b) Crown damage in % caused by R. solani (perimeter affected); c) % total damage of roots caused by the complex F. oxysporum f. sp. lycopersici - R. solani - P. lycopersici; d) Total yield; e) First quality fruits. Results were analysed using ANOVA and Duncan's tests at p<0.05. Percent values were transformed into Bliss degree as described (Rustom et al. 1989) for the statistic analysis. The
determination of the inoculum of phytopathogens found in soils was
done as follows: soil samples were taken up to Th650 was formulated as alginate pellets, as described (Montealegre and Larenas, 1997), reaching a concentration of 4.2 x 106 cfu/g pellets in assay 1 (winter) and of 570.000 cfu/g pellets in assay 2 (summer). Pl629 was formulated as described (Raupach and Kloepper, 1998). Characterisation of biocontrollers Th650 grown in the presence of cell walls from the different phytopathogens of the complex showed: a) secretion of two chitinases, one β-1,3-glucanase and a wide band of protease activity in response to cell walls of F. oxysporum sp. lycopersici, b) secretion of one chitinase, three β-1,3-glucanases and high protease activity in response to cell walls of P. lycopersici, and c) two chitinases, four β-1,3-glucanases and high protease activity in response to cell walls of R. solani (Figure 1). Therefore, Th650 has the ability to secrete three different types of hydrolytic enzymes involved in biocontrol against the three phytopathogens tested. The differences observed both in isoenzymic patterns and levels of enzyme activity could reflect the ability to induce the expression of specific genes for the degradation of the polysaccharides found in the cell walls from the different phytopathogens tested, in response to the presence of their cell walls (Pérez et al. 2002). In fact, Th650 expressed and secreted two endochitinases towards R. solani and F. oxysporum one endochitinase towards P. lycopersici; four β-1,3-glucanase isoenzymes in response to the presence of cell wallsfrom R. solani, three in response to P. lycopersici and only one in to F. oxysporum (Figure 1).Also, the highest endochitinase and β-1,3-glucanase activities were observed in supernatants of Th650 cultures with R. solani cell walls as the sole carbon source. The secretion of chitinolytic enzymes and β-1,3-glucanases has been detected in the rhizosphere of soybean seedlings inoculated with T. harzianum and planted in a soil infested with R. solani demonstrating that T. harzianum was the source of these enzymes in response to the presence of R. solani (dal Soglio et al. 1998). These enzyme activities have been also detected in supernatants from other T. harzianum isolates in response to R. solani or F. oxysporum or P. lycopersici cell walls (Pérez et al. 2002), although isoenzymic patterns differ among isolates. The secretion of difusible and volatile antibiotics, previously demonstrated for Th650 (Escobar et al. 2004), along with the secretion of biocontrol enzymes, indicate that Th650 has the ability to use different biocontrol mechanisms (Benítez et al. 2004) against the phytopathogens already mentioned. On the contrary, the secretion of antibiotics appears to be the most probable biocontrol mechanism of Pl629, similar to other antagonistic bacteria (Guo et al. 2004), because it is unable to secrete any of the biocontrol enzymes mentioned above (Montealegre et al. 2003). As opposed to Pl629, Serratia or Bacillus cereus include the secretion of chitinases within their biocontrol mechanisms (Ordentlich et al. 1988; Frankowski et al. 2001; Huang et al. 2005). From this point of view, the combined use of Th650 plus Pl629 would cover a wide spectrum of antagonistic mechanisms. Innoquity test of Th650 on tomato plants No lesions were observed in roots or in crowns of tomato plants, neither seed germination was affected by the presence of Th650, suggesting that this Trichoderma isolate is not pathogenic for tomato seeds or seedlings. A 100% emergence at day 7 was observed for all seeds, and no significant differences were observed related to mortality, dry weight of roots or aerial portion, crown diameter and seedling height. Based on these results, any negative effect of Th650 was discarded. Control
of root damage. The ability of Th650 and of Pl629 to decrease root
damage caused either by P. lycopersici or by the complex of
pathogens in the winter assay, is shown in Table 1. Analysis of soils
showed that they contained 1.6 to 2.0 x 104 cfu/g soil
of P. lycopersici, 5.3-6.3 x 104 cfu/g soil of F.
oxysporum and 1.72-2.27 x 105 cfu/g soil of R. solani,
which is considered a high inoculum. Treatments which included MeBr
alone or MeBr plus either Th650 or Pl629 showed significant differences
in reducing % root damage caused by P. lycopersici when compared
to control; although no differences were observed among these three
treatments suggesting that the effect is mainly due to MeBr. It was
expected no root lesion in treatment with MeBr, but a 2.2 damage index
was observed suggesting that re-colonisation of soil by P. lycopersici
could have been produced. This agrees with previous studies that showed
that re-colonisation occurred because of the biological void produced
as a consequence of MeBr treatment (Montealegre et al.
1996). As the decrease in % root damage showed no significant
differences when compared to MeBr alone, it is possible that none
of the bioantagonists could prevent the re-colonisation by P. lycopersici.
These results agree with the fact that BL629 does not secrete any
enzyme system involved in bio-control, and that Th650 express only
one chitinase with very low activity in response to the presence of
this phytopathogen, which could be insufficient to control the development
of P. lycopersici (Figure 1). Therefore,
it appears that chitinases secreted by bio-controllers are important
in preventing P. lycopersici development as well as tomato
root damage, which agrees with the fact that a different T. harzianum
isolate (isolate 11) accomplished an effective control of P. lycopersici,
that correlates with a high production of chitinase activity expressed
as multiple isoforms (Pérez et al. 2002). It is
also important to mention that the % root damage observed in the presence
of Th650 or of Pl629 alone or in combination among them, did not show
significant differences from the control, suggesting that none of
the antagonists could prevent the damage caused by P. lycopersici,
as opposite to the findings of in vitro experiments (Pérez
et al. 2002). In addition, it must be taken into account that
according to optimal temperature development, P. lycopersici
is the main pathogen found during winter season (Jones
et al. 1991). In this season its development is higher than the
one observed for F. oxysporum f. sp. lycopersici but
similar to that of R. solani. On the other hand, optimal development
of Th650 is at The % root damage produced by the presence of the complex of pathogens was significantly different when control was compared to treatments that contained MeBr. The treatment with this latter compound was not improved by the addition of Pl629 or by Th650, suggesting that the decrease in root damage was due to the presence of MeBr rather than to the presence of the bacterial or the fungal antagonist. As in the previous case, the advantage in development of P. lycopersici and R. solani in the winter season could explain results in terms of root damage. Based on the results obtained in this winter assay, we decided to omit treatments that included the combined use of Th650 and Pl629, MeBr and Th650 and MeBr and Pl629 in the summer assay. Fruit yield and quality. When this assay was evaluated in terms of yield and fruit quality, significant differences were observed among treatments that included MeBr, or Th650 and control in terms of total yield per plant in the winter season (Table 1). In these conditions, Th650 was as good as MeBr alone, suggesting that although it is not controlling root damage it is improving yield as compared to control. This could be due to a yield promoting activity of Trichoderma species, similar to that described for a growth promoting activity of these fungi (Baker et al. 1984). Combination of this fungus and MeBr did not improve statistically the effect of any of them. Replacement of Th650 by Pl 629 neither improved total yield. First quality fruits produced after any of the treatments were not different from controls, with the exception of MeBr + Th650 or Pl629. These results could be explained as a result of the growth promoting activity of Trichoderma and Paenebacillus (Benítez et al. 2004; Guo et al. 2004). Finally, the presence of these bio-controllers is not affecting the % of first quality fruits. Control of root damage.When the assay was performed in the summer season, we could include solarization in addition to some of the treatments performed in winter. Percent root damage caused by P. lycopersici or R. solani or the complex is shown in Table 2. Root
damage of tomato plants caused by P. lycopersici in the summer
season was similar to that observed in the winter season assay. Treatment
with MeBr resulted in less root damage being also the most effective
treatment, suggesting that fumigation with MeBr was favoured by the
soil temperature. Solarization also reduced significantly root damage
caused by this pathogen. During solarization, soil temperature reached
a mean of As in the winter season assay, Th650 did not show any biocontrol activity against P. lycopersici although the soil temperature during the summer assay was not limiting fungal development. Pl629 alone behaved as Th650 alone, and combination of both bio-controllers with solarization did not improve the effect of the latter treatment. Taking these results altogether, it appears that the lack of biocontrol of Th650 and of Pl629 is not due to a seasonal effect but to their lack of capacity to express effective biocontrol mechanisms on P. lycopersici. On the contrary, both Th650 and Pl629 prevented root damage caused by R. solani when applied alone or after solarization. Expression of chitinases, β-1,3-glucanases and proteases by Th650 in terms of number of isoenzymes and level of activity (Figure 1) is clearly better towards R. solani, fact that could account for the ability of Th650 to control root damage produced by this pathogen. On the other hand, although Pl629 does not secrete these enzyme systems, it can produce antibiotics that may be affecting R. solani development, or improving root development as has been described for other growth-promoting bacteria (Guo et al. 2004). Solarization alone significantly decreased root damage produced by R. solani, and when combined with Pl629 showed better results than the treatment alone. Therefore, it may be suggested that this bacterium might be controlling the re-colonisation of soil by R. solani after solarization, as opposite to what occurs for P. lycopersici. The addition of Th650, although decreased the % of total damage, was not significantly different from solarization alone. Finally, although fumigation with MeBr was statistically different from solarization alone, when Th650 or Pl629 were used in combination with solarization, results were comparable to those obtained with MeBr, thus constituting good alternatives to the use of MeBr for the control of R. solani. The % root damage caused by the complex in the summer season was significantly reduced by MeBr (Table 2) as was observed in the winter season (Table 1). The summer assay also showed that solarization was effective in controlling root damage produced by the complex, but its effect could not be compared to the use of MeBr. The weakening and delayed mortality of F. oxysporum already described (Assaraf et al. 2002) could be contributing to control the root damage already mentioned. The use of Th650 or of Pl629 decreased % total damage, effect that could be attributed to an effective action of the bio-controllers on R. solani. Th650 was less efficient than solarization, and Pl629 showed no significant differences with solarization in reducing % root damage. The combined use of Pl629 or Th650 with solarization did not improve the effect of the biocontrollers or solarization alone. Fruit yield and quality. The treatments used for the summer season assay did not result in significant differences either in fruit yield or in fruit quality (Table 2). Moreover, MeBr alone was not better than the other treatments when total yield or first fruit quality were evaluated. Taken together the results of both assays, it may be concluded that Th650 and Pl629 do not express bio-control mechanisms enough to prevent P. lycopersici development, but appear to be good alternatives to the use of MeBr for the control of R. solani. Solarization prevents root damage due to the presence of P. lycopersici or R. solani or of the complex; nevertheless, this prevention did not result in improvement of fruit yield or quality. In addition, solarization could be improved by the addition of Th650 or Pl629 for the control of R. solani. ASSARAF, M.; GRINZBURG, C. and KATAN, J. Weakening and delayed mortality of Fusarium oxysporum by heat-treatment: flow citometry and growth studies. Phytopathology, 2002, vol. 92, no. 9, p. 956-963. BAKER, R.; ELAD, Y. and CHET, I. The controlled experiment in the scientific method with special emphasis in biological control. Phytopathology, 1984, vol. 74, p. 1019-1021. BENÍTEZ, T.; RINCÓN, A.M.; LIMÓN, M.C. and CODÓN, A.C. Biocontrol mechanisms of Trichoderma strains. International Microbiology, 2004, vol. 7, no. 4, p. 249-260. CAMPBELL, R. and SHISHKOFF, N. Survival of Pyrenochaeta lycopersici and the influence of temperature in cultivar resistance on the development of corky root of tomato. Plant Disease, 1990, vol. 74, no. 11, p. 889-894. CHET,
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