The Protective Effect of Trichoderma asperellum on Tomato Plants against Fusarium oxysporum and Botrytis cinerea Diseases Involves Inhibition of Reactive Oxygen Species Production

Trichoderma species are fungi widely employed as plant-growth-promoting agents and for biological control. Several commercial and laboratory-made solid formulations for mass production of Trichoderma have been reported. In this study, we evaluated a solid kaolin-based formulation to promote the absortion/retention of Trichoderma asperellum in the substrate for growing tomato plants. The unique implementation of this solid formulation resulted in an increased growth of the tomato plants, both in roots and shoots after 40 days of its application. Plants were challenged with two fungal pathogens, Fusarium oxysporum and Botrytis cinerea, and pretreatment with T. asperellum resulted in less severe wilting and stunting symptoms than non-treated plants. Treatment with T. asperellum formulation inhibited Reactive Oxygen Species (ROS) production in response to the pathogens in comparison to plants that were only challenged with both pathogens. These results suggest that decrease in ROS levels contribute to the protective effects exerted by T. asperellum in tomato.


Introduction
One goal of modern agriculture is the production of safe food through sustainable and eco-friendly practices. These involve a cutback in the use of chemicals in the fields to reduce potential environmental damage. In this regard, biological agents constitute an excellent alternative to replace chemicals for pest control or growth improvement. Trichoderma species are widely used for field application as biofungicides against pathogens such as Botrytis cinerea, Fusarium spp., Pythium spp., Rhizoctonia solani and Sclerotium rolfsii on crops of economic importance [1,2].
Trichoderma is a ubiquitous genus of filamentous fungi that growths in the rhizosphere and colonize plant roots as an opportunistic, avirulent plant symbiont [3]. Trichoderma spp. receives nutrients from root exudates in exchange for plant protection against biotic and abiotic stresses [4]. The interface between Trichoderma and plant is established by early events that include the release of elicitors from Moreover, we found that T. asperellum prevented ROS generation. These low ROS levels were associated with disease control promoted by T. asperellum.

Kaolin Formulation Allows Trichoderma Asperellum High Biomass, Viability and Stability
Trichoderma produces large amounts of fungal spores and this characteristic makes it ideal for inoculum production under laboratory conditions. In this study, we used a solid formulation devised to promote Trichoderma proliferation and survival in soil. The solid formulation consisted of an oat flakes/kaolin-vermiculite mixture that functions as an inexpensive, simple and readily available source of nutrients.
To evaluate T. asperellum spore viability in the growing substrate, CFUs were quantified several days after application. As shown in Figure 1, approximately 1 × 10 7 CFU/g soil remained at 4 dpi, and those levels were maintained throughout the experiment (40 days), indicating an optimal retention and survival of T. asperellum in the solid matrix. Moreover, this formulation showed a ten-fold higher biomass production of Trichoderma inoculum than the bentonite-vermiculite formulation employed with T. harzianum [31]. A possible explanation for this logarithmical difference may be due to the formulation pH. According to Singh [33], T. asperellum produces high biomass at pH 7.0 with a slow preference at pH 5.5. In our study, the pH recorded in the kaolin was 5.9, suggesting that an acidic milieu positively influences spore production.

Kaolin Formulation Allows Trichoderma Asperellum High Biomass, Viability and Stability
Trichoderma produces large amounts of fungal spores and this characteristic makes it ideal for inoculum production under laboratory conditions. In this study, we used a solid formulation devised to promote Trichoderma proliferation and survival in soil. The solid formulation consisted of an oat flakes/kaolin-vermiculite mixture that functions as an inexpensive, simple and readily available source of nutrients.
To evaluate T. asperellum spore viability in the growing substrate, CFUs were quantified several days after application. As shown in Figure 1, approximately 1 × 10 7 CFU/g soil remained at 4 dpi, and those levels were maintained throughout the experiment (40 days), indicating an optimal retention and survival of T. asperellum in the solid matrix. Moreover, this formulation showed a ten-fold higher biomass production of Trichoderma inoculum than the bentonite-vermiculite formulation employed with T. harzianum [31]. A possible explanation for this logarithmical difference may be due to the formulation pH. According to Singh [33], T. asperellum produces high biomass at pH 7.0 with a slow preference at pH 5.5. In our study, the pH recorded in the kaolin was 5.9, suggesting that an acidic milieu positively influences spore production.

Trichoderma Asperellum Stimulates Plant Growth on Tomato Plants
To characterize the effect of T. asperellum on plant growth and development, two-week-old tomato seedlings were grown in sterile soil or in T. asperellum-treated soil mixes. After 40 days, growth parameters were measured. Plant absolute growth rate and chlorophyll content were 22% and 16% higher, respectively in T. asperellum-treated plants than in control plants (Table 1). Both fresh and dry weight were 30% higher in treated plants, as well. Leaf area was not significantly different between the two treatments (Table 1).

Figure 1.
Trichoderma asperellum viability in the growing media. T. asperellum CFU per gram was quantified at various times after mixing with the growing substrate. The population was expressed as Log CFU/gram-Bars indicated SD from three independent experiments.

Trichoderma Asperellum Stimulates Plant Growth on Tomato Plants
To characterize the effect of T. asperellum on plant growth and development, two-week-old tomato seedlings were grown in sterile soil or in T. asperellum-treated soil mixes. After 40 days, growth parameters were measured. Plant absolute growth rate and chlorophyll content were 22% and 16% higher, respectively in T. asperellum-treated plants than in control plants (Table 1). Both fresh and dry weight were 30% higher in treated plants, as well. Leaf area was not significantly different between the two treatments (Table 1). Data are means ± S.D. from ten biological replicates. Same letter in the column denote no significant difference between both treatments according a pairwise comparison using Student's t-test (p < 0.05). * Based on plant height.

Trichoderma Asperellum Reduces Fusarium Oxysporum Wilt Symptoms in Tomato Plants
The effectiveness of T. asperellum as a biocontrol agent was evaluated by recording the disease symptoms induced by F. oxysporum ( Figure 2). Two-week-old plants were planted in the T. asperellum-solid formulation or mock, and three weeks after were inoculated with F. oxysporum. The plants were grown for three weeks post-inoculation (wpi) before disease symptoms assessment. Less severe disease symptoms caused by F. oxysporum were recorded in T. asperellum-inoculated plants than in untreated plants. Symptoms included wilting and stunting ( Figure 2A). This result was concordant with the reduction in fresh-weight loss triggered by fungal pathogen infection in T. asperellum-inoculated plants in reference to non-pretreated plants ( Figure 2B).
Disease severity was estimated according to leaf damage established by the scale shown in Figure 2C (upper panel) for plants after one and three weeks of infection with Fusarium. While plants infected with F. oxysporum mainly exhibited the most severe symptoms, after 3 weeks, T. asperellum-treated plants and infected with F. oxysporum displayed disease severity values averaging between one and two. These results support the role of T. asperellum as a successful biocontrol agent against Fusarium wilt.
F. oxysporum plant colonization was analyzed by cutting stem sections from different nodes of non-and pretreated plants and plated on PDA [34].
As it is shown in Figure 2D, F. oxysporum colonized cotyledon node and first node of mock plants. In a similar way, F. oxysporum was detected in the same nodes of T. asperellum-pretreated plants. However, the biocontrol agent showed faster growth. This result contrasts with the report by Martínez-Medina et al. [32] who did not detect Trichoderma presence in the shoots of tomato plants inoculated in the roots. In this regard, a possible contamination cannot be ruled out but neither that T. asperellum can be translocated through the vascular system.

Trichoderma Asperellum Induces Systemic Resistance against Botrytis Cinerea
To investigate the protective systemic effects of T. asperellum against fungal disease, detached leaves from mock and pretreated T. asperellum plants were inoculated with B. cinerea. Lesion development was recorded two weeks after infection. Detached leaves from T. asperellum-pretreated plants showed lower and less severe lesions compared to non-treated plants ( Figure 3A,B). These results are in accordance with Martínez-Medina [32], who found a 30% decrease in lesion size in plants inoculated with T. harzianum and infected by B. cinerea. non-and pretreated plants and plated on PDA [34].
As it is shown in Figure 2D, F. oxysporum colonized cotyledon node and first node of mock plants. In a similar way, F. oxysporum was detected in the same nodes of T. asperellum-pretreated plants. However, the biocontrol agent showed faster growth. This result contrasts with the report by Martínez-Medina et al. [32] who did not detect Trichoderma presence in the shoots of tomato plants inoculated in the roots. In this regard, a possible contamination cannot be ruled out but neither that T. asperellum can be translocated through the vascular system.

Trichoderma Asperellum Induces Systemic Resistance against Botrytis Cinerea
To investigate the protective systemic effects of T. asperellum against fungal disease, detached leaves from mock and pretreated T. asperellum plants were inoculated with B. cinerea. Lesion development was recorded two weeks after infection. Detached leaves from T. asperellum-pretreated plants showed lower and less severe lesions compared to non-treated plants ( Figure 3A, 3B). These results are in accordance with Martínez-Medina [32], who found a 30% decrease in lesion size in plants inoculated with T. harzianum and infected by B. cinerea.

Trichoderma Asperellum Supresses ROS Production against Necrotrophic Pathogens
ROS production is an early event during plant defense responses triggered by pathogens attack. In this context, the effect of T. asperellum on ROS accumulation was investigated in tomato leaves infected with F. oxysporum and B. cinerea in the first 24 h ( Figure 4A,B, respectively). ROS production was recorded three weeks after inoculation of tomato plants with T. asperellum.

Trichoderma Asperellum Supresses ROS Production against Necrotrophic Pathogens
ROS production is an early event during plant defense responses triggered by pathogens attack. In this context, the effect of T. asperellum on ROS accumulation was investigated in tomato leaves infected with F. oxysporum and B. cinerea in the first 24 h ( Figure 4A and 4B, respectively). ROS production was recorded three weeks after inoculation of tomato plants with T. asperellum. As Figure 4 shows, ROS production was markedly diminished in tomato plants preinoculated with T. asperellum in comparison to non-treated plants infected with both fungal pathogens. ROS production was detected at 3 hpi, and it was sustained until 24 h and 12 h in plants infected with F. oxysporum and B. cinerea, respectively.

Discussion
We found that an oat/kaolin-vermiculite-based formulation for T. asperellum application was highly effective for sustaining the biocontrol agent and to protect tomato plants from F. oxysporum As Figure 4 shows, ROS production was markedly diminished in tomato plants preinoculated with T. asperellum in comparison to non-treated plants infected with both fungal pathogens. ROS production was detected at 3 hpi, and it was sustained until 24 h and 12 h in plants infected with F. oxysporum and B. cinerea, respectively.

Discussion
We found that an oat/kaolin-vermiculite-based formulation for T. asperellum application was highly effective for sustaining the biocontrol agent and to protect tomato plants from F. oxysporum and B. cinerea. This protective effect was associated with the reduction of ROS production in response to pathogen infection. Under these conditions, where grinding or extrusion of bioformulation were not considered, T. asperellum viable biomass, determined as CFUs, remained unchanged over 40 days and no contamination was detected in the culture examinations, indicating that kaolin was not a source of contaminants, as it was previously reported [12]. Moreover, T. asperellum retained its physiological and biochemical activities in the solid formulation since its application to tomato plants led to enhanced growth and tolerance against pathogens.
Many reports have shown that Trichoderma spp. improves the growth in many plant species [11]. In this study, we demonstrated that T. asperellum improved plant growth parameters including plant absolute growth rate, and fresh and dry weight with a significant difference relative to mock treatment, except leaf size. It is important to highlight the enhancement of shoot and root growth as it has been reported [35,36].
Chlorophyll content was also increased in T. asperellum-treated plants as was observed in melon and cacao plants inoculated with T. harzianum and T. asperellum, respectively [31,37]. This result suggests an optimal physiological status of plants.
Besides its plant growth stimulatory activity, Trichoderma spp. are fungi that reduce negative effects by plant pathogens. In our study, we investigated the suppressive role of T. asperellum against two fungal pathogens of tomato plants. T. asperellum reduced disease symptoms development upon infection or F. oxysporum or B. cinerea in tomato plants.
It has recently been reported that SA and JA are involved in T. virens-mediated resistance in tomato against F. oxysporum. Jasmonic acid-deficient def-1 and salicylic acid-deficient NahG mutants are more susceptible to infection. Measurement of JA in wild type plants revealed that T. virens cultured on barley grains or the combination T. virens plus F. oxysporum showed higher JA levels and lower disease incidence. By comparison, F. oxysporum-treated plants only showed a small increase in the JA levels [38]. These results suggest that T. virens is the responsible agent to induce JA changes in the plant like a defense mechanism. In contrast, SA content increased in response to the pathogen. However, SA was only slightly built up in plants treated with T. virens cultured on barley grains or in the combination of T. virens plus F. oxysporum [38]. The involvement of JA and SA has also been demonstrated in the resistance mediated by T. harzianum of tomato plants against B. cinerea. The resistance promoted by T. harzianum is also lost in def-1 and NahG mutants. In addition, def-1 mutants showed higher colonization of B. cinerea in comparison of wild type infected plants [32], suggesting that JA is controlling the pathogen proliferation in the plant tissues.
The protection mediated by T. asperellum against both pathogenic infections could be related to growth promotion, at least in part. However, in this study we report that T. asperellum inhibited ROS the common production upon pathogen infection. To avoid oxidative stress, the cells use a radical scavenging mechanism to neutralize free radicals or reactive species. Superoxide dismutase (SOD) is an enzyme whose activity is involved in the earliest defense responses. It is considered the first detoxifying line. SOD catalyzes the dismutation of superoxide anion to hydrogen peroxide [39]. T. harzianum has been shown to increase SOD activity in tomato plants after F. oxysporum infection [40]. Nevertheless, we only detected a weak H 2 O 2 accumulation in the tomato leaves of plants pretreated with T.asperellum, which were subsequently pathogen-infected with reference to unpretreated plants challenged with the pathogen. According to Zehra et al. [40], the pretreatment of tomato plants with T. harzianum supresses ROS builds up by enhancing mechanisms such as antioxidant defense mediated by catalase and ascorbate peroxidase activities in response to F. oxysporum infection.
B. cinerea secretes toxins to kill plant cells. The most known are botrydial and its by-products and botcinic acid derivatives [41,42]. Besides these toxins, B. cinerea itself produces ROS in the plant during the infection process as a virulence factor. H 2 O 2 accumulates in the early steps of infection, both in germinating spores and in the infection cushions [43]. B. cinerea shows resistance against the oxidative burst induced as a plant-host defense response, protecting itself with an extracellular catalase activity [44]. Beyond survival to an oxidative environment during plant-pathogen interaction, B. cinerea also stimulates ROS production in the plant. Early production of ROS and host cell death are indicative of a successful infection by B. cinerea [45]. In some plant-pathogen interactions, PCD promotes pathogen growth, especially those pathogens that secret toxins to the host cells [46]. ROS increase in Arabidopsis enhances necrosis induced by B. cinerea and this necrosis is dependent on host-activated HR and not by fungus toxicity, since dnd1, a mutant with HR-deficiency is asymptomatic in response to B. cinerea infection [47]. Moreover, B. cinerea growth is fostered by ROS generation and HR establishment. The oxidative burst is effective to contend with biotrophic pathogens, but it is unable to protect the host against necrotrophs [48]. The above results demonstrated a correlation between ROS and lesion formation in response to B. cinerea infection in tomato plants, as was previously reported in Arabidopsis [47]. Massive necrosis was observed in plants infected with B. cinerea without T. asperellum pre-treatment. Under this condition, Botrytis lesion formation increased two-fold as compared to plants pretreated with T. asperellum. Regarding H 2 O 2 production, this was suppressed at all times as evaluated by T. asperellum pretreatment suggesting that the biocontrol agent promoted resistance by inhibiting ROS formation and HR-like lesions.
A possible explanation for the mechanism by which Trichoderma spp mediates resistance in response to F. oxysporum and B. cinerea is through JA involvement, since this hormone controls fungi proliferation, up-regulates the expression of defense genes, induces the activities of catalase and ascorbate peroxidase and shows minimal lipid peroxidation and cell death after a challenge infection with these pathogens. Further research is required to elucidate the JA participation in the resistance induced by Trichoderma spp.

Plant Material
Lycopersicon esculentum genotype Vita was used in the experiments. Seeds were surface-sterilized in 0.5% sodium hypoclorite for 5 min, and rinsed twice with sterile water for 10 min before use. Seeds were germinated on 200-cavity trays with sterile peat moss and perlite (agrolite). Seedlings were grown in a greenhouse under 14 h light and 10 dark photoperiod at 27 • C and 12 • C, respectively.

Trichoderma Inoculum Preparation
The bio-agent was obtained from Dr. Hernández at Centro de Biotecnología Genómica (IPN), México. A pure culture of T. asperellum was maintained on PDA plates at 25 • C for 7 days. For inoculum preparation, a solid formulation (20 g of oat, 50 mL of kaolin, 100 mL of vermiculite and 60 mL of water (modified from Martínez-Medina et al. [31]) was sterilized and the substrate was later inoculated with T. asperellum (1 × 10 5 spores mL −1 ) under sterile conditions and incubated for one week at 28 • C.

Inoculation of Tomato Plants with T. Asperellum
After complete plant emergence at 2 weeks, individual seedlings were thinned out to 6-inch pots containing commercial peat moss and vermiculite (7:3, v/v) supplemented with a mixture containing the solid substrate previously inoculated with T. asperellum or mock. Plants were returned to the green house.

Quantification of T. Asperellum in Growing Media
To quantify T. asperellum in the growing media, one gram of soil mixture containing the solid substrate inoculated with T. asperellum or mock was plated on potato dextrose agar (PDA) supplemented with 50 mg L −1 rose bengal and 10% streptomycin sulphate [31]. Medium was pH 4.9 adjusted with lactic acid. Plates were incubated at 28 • C for 5 days and then colony-forming units (CFUs) were counted. Data were expressed as Log CFU per gram of dry substrate.

Evaluation of Tomato Plant in Growing Media Inoculated with T. Asperellum
Plant Absolute Growth Rate (AGR) was evaluated forty days after inoculation with. T asperellum by recording shoot height. AGR was calculated using the formula AGR = (H2 − H1)/(t2 − t1) [49]. Leaf area (cm 2 ), chlorophyll content, fresh weight (g) and dry weight (g) were also evaluated at 40 days post inoculation. For leaf area, individual leaves of tomato plants were scanned and subsequently analyzed by Image J 1.44 [50]. Optical measurements were made on leaves of 10 plants using a SPAD-502Plus (Konica Minolta Sensing, Inc., Tokyo, Japan). Fresh and dry weight were measured with a precision analytical balance. All measurements were recorded for at least 10 plants per treatment with three independent repetitions.

Isolation and Identification of Fungal Pathogens
A Fusarium strain was isolated from rotted tomato (cv. Saladette) roots by placing the disinfected tissue on PDA plates and incubating for 7 days. The mycelia emerging from the tissue was transferred to a fresh PDA plate and a mycelial plug from this plate was resuspended in sterile water. Serial dilutions from these spore suspensions were performed and a 100-µL aliquot of a 100-fold dilution was plated on 1% agar and incubated for 24-48 h. Single-conidia germinating were transferred to PDA plates and an agar plug from these cultures was used to inoculate GYAM liquid media. The mycelial mat from 5-day-old culture was used to extract genomic DNA. The PCR reaction was performed for 30 cycles using the ef1 (forward primer: 5 -ATGGGTAAGGA(A/G)GACAAGAC-3 ) and ef2 (reverse primer: 5 -GGA(G/A)GTACCAGT(G/C)ATCATGTT-3 ) with an annealing temperature of 53 • C. The 700-bp amplicon was gel-purified and used for DNA sequencing with the ef22 internal reverse primer (5 -AGGAACCCTTACCGAGCTC-3 ), according to Geiser et al. [51]. The 350-bp sequence was analyzed through the Fusarium ID platform (http://isolate.fusariumdb.org/) for species identification.
B. cinerea was isolated on PDA media from tomato fruits showing typical signs of grey mould. Plates were incubated at 22 • C with 14 h darkness and 10 h light for 7 days. Pathogenicity tests were performed following Koch's postulates.

Evaluation of Effectiveness of T. asperellum against Fungal Pathogens in Tomato Plants
Roots of plants grown for 3 weeks in media with and without T. asperellum formulation were cut about 1 cm from the tips, and the roots were placed during 5 min in the F. oxysporum suspension (1 × 10 5 conidia mL −1 ). Control plants were mock-inoculated with sterile distilled water. Disease progression was followed through photograph records of symptoms 3 weeks post-inoculation (wpi). Plant fresh weight was recorded at 1 and 3 wpi.
Disease severity was calculated by the number of leaves showing different levels of wilt symptom and expressed as percentage in reference of total number of leaves per plant. Symptoms were recorded at 1 and 3 wpi. Phenotypic analysis, disease severity and loss weight assays were repeated three times with 10 replicates per treatment.
To explore disease progression, a colonization assay was performed in non-and pretreated plants with T. asperellum for 3 weeks and infected with F. oxysporum for 3 weeks.
Stem sections were collected from cotyledon node, first and second nodes. Sections were surface sterilized in 70% ethanol and rinsed in sterile distilled water and placed on PDA supplemented with 200 mg/mL streptomycin at 25 • C. Photographs were taken after 5 days of incubation on PDA. The experiment was repeated three times.
The B. cinerea strain was routinely cultivated in Petri dishes containing PDA. For inoculation, detached leaves from 5-week-old plants were laid on Petri dishes containing two blotting filter papers (Whatman) wetted with sterile water, then spotted with 5-mm-diameter agar plugs containing growing hyphae from B. cinerea [52]. Lesions were measured using the ImageJ software employing a calibration scale [50] at 1 and 2 wpi. Experiments were repeated three times with 10 replicates per treatment.

Evaluation of T. asperellum Effect on H 2 O 2 Production in Tomato Fungal-Infected Plants
DAB is taken up by living plant tissue and can be used to show H 2 O 2 production when peroxidase activity is present [53]. Detection of hydrogen peroxide was conducted using 3,3 -diaminobenzidine (DAB) from Sigma-Aldrich. Leaves were infected with F. oxysporum or B. cinerea, respectively and collected at different times. Plant tissues were immersed with 1 mL of DAB liquid buffer solution for 2 h. After staining, the tissues were fixed in ethanol:glycerol:acetic acid 3:1:1 (v:v:v) (bleaching solution) placed in a water bath at 95 • C for 15 min. Leaves were reimmersed in bleaching solution until chlorophyll was completely depleted.

Statistical Analyses
Data on AGR, leaf areas, SPAD units, fresh and dry weight and disease severity were subjected to a pairwise comparison with Student's t-test (p < 0.05) implemented in Excel software.

Conclusions
This study demonstrates the successful use of a solid formulation based in kaolin as inoculum with T. asperellum to enhance tomato plants growth and defense against pathogens. The fact that T. asperellum inhibits ROS accumulation makes it an excellent candidate to control fungal pathogens that promote oxidative stress as mechanism for successful disease establishment. The application of this bioformulation can have a positive impact on disease management in sustainable agriculture. The effective application of the bioformulation in the field requires more research on issues such as shelf life of the product and its viability during storage.