Defense response by interactive bio-protector and chitosan to Sclerotium rolfsii Wilt disease on cowpea , Brazilian Oxisol

Pathogenic microorganisms increase enzyme production and plant response, causing injuries and even plant death. Chitosan has shown potential to induce reactions in plant pathogens. An “in vitro” assay determines the action of chitosan and evaluates the optimal concentration to act against (Sclerotium rolfsii). The anti-microbial activity of chitosan and the bio-protector produced by the inter-active bacteria Beijerinckia indica and fungus Caenorhabditis elegans that contain chitosan were tested in a pot experiment against S. rolfsii on cowpea grown in a Brazilian Oxisol. Biofertilizer and chitosan in foliar application were used with and without S. rolfsii. The plants were analyzed, and the disease severity index, soluble protein and enzymatic activities were determined. The “in vitro” test showed chitosan effectiveness against S. rolfsii. The pot experiment with sterilized and non-sterilized soil confirmed the ability of the bio-protector that contains chitosan from C. elegans and chitosan application to increase enzymatic activities processed by S. rolfsii. The higher concentrations of the bio-protector and chitosan (4.0 and 6.0 mg mL -1 ) were directly related to the catalase and peroxidase activities controlling plant resistance and the disease severity index. The bio-protector may be a viable alternative to soluble fertilizer, and recommended for organic and sustainable agriculture. These findings are important for the establishment of sustainable agriculture and to avoid the use of pesticides.


INTRODUCTION
Cowpea (Vigna unguiculata L. Walp) is a legume of socioeconomic importance in tropical countries, especially in Brazil, because it represents a popular dietary source of protein, carbohydrates, iron, potassium and phosphorus.Cowpea legumes can be used for animal feed, forage, hay, silage, flour and soil fertility as green manure; additionally, it has considerable tolerance to drought and heat stresses (Berger et al., 2016).
Sclerotium rolfsii (Sacc) is one of the most important phytopathogenic fungi in the tropical and sub-tropical regions, and it causes serious damage in more than 500 plant species, including agricultural crops; in the cowpea legume, it causes a frequent and important disease known as Sclerotium Wilt disease, according to Sharma et al. (2012).This pathogen affects the soil and can grow and survive for long periods in the form of chlamydospores, and the symptoms of the disease in cowpea reduce the plant growth and cause chlorosis, wilting and premature leaf fall, which almost always result in the death of infected plants (Berger et al., 2016).
Normally, the application of large amounts of chemical products (fungicides) is necessary to control plant diseases, which causes serious problems in the soil ecosystem.Natural substances studied as alternatives to control pathogenic microorganisms reduce the injuries; however, it is well-known that these products are even prejudicial to the environment and may promote food contamination (Thakkar and Sarafi, 2015).
Chitin is a natural biopolymer of N-acetyl-dglucosamine that is present as structural elements in invertebrates, in most arthropods and in the cell walls of fungi, especially from the Mucorales Order and Zygomycetes classes (Bautista-Baños et al., 2016).Chitosan is a biodegradable and biocompatible polysaccharide that has antifungal activity, inhibits the growth of pathogenic fungi and induces defense mechanisms in plants.Chitosan also stimulates the accumulation of proteins and the production of reactive oxygen species, alters the metabolism of phytoalexins, induces the formation of phenolic compounds, and activates peroxidase, chitinase, β-1,3-glucanase, superoxide dismutase and catalase enzymes (Andrade et al., 2013).
The correct use of chitosan promotes a large increase in the activity of catalase and peroxidase, and these enzymes are involved in plant defense against pathogenic microorganisms, according to Dousseau et al. (2016).In recent studies, the biopolymer chitosan demonstrated antimicrobial activity against pathogenic fungi and the ability to induce mechanisms of plant defense (Romanazzi et al., 2013).
The aim of this study is to evaluate the defense response to S. rolfsii by the bioprotector produced from phosphate and potassic rocks mixed with organic matter inoculated with interactive diazotrophic bacteria (Beijerinckia indica) and Cunninghamella elegans (Mucorales fungi) on cowpea in a Brazilian Oxisol of the rain forest region.

Test for chitosan: In vitro assay
An in vitro assay was conducted in Petri dishes to test the effectiveness of crustaceous chitosan applied in various concentrations against the pathogenic fungus S. rolfsii.The phytopatogenic fungus S. rolfsii used in the present study causes Sclerotium Wilt in cowpea, which interferes with important oxidative damage.The fungus was isolated in the laboratory from inoculated plants that presented symptoms of the disease in cowpea seedlings, transferred to Petri dishes containing potato-dextroseagar (PDA) medium, and maintained for 15 days in a camera biochemical oxygen demand (BOD) at 28°C.The fungus pathogenicity was confirmed by characteristics of the culture when inoculated on cowpea seedlings (Berger et al., 2016).
To evaluate the mycelia growth of the S. rolfsii isolate, the crustaceous chitosan, purchased at a medium molecular weight from Sigma-Aldrich Chemical Company, was used.To obtain the gel chitosan, the biopolymer was mixed for 24 h in a horizontal shaker and then incorporated into potato-dextrose-agar medium, obtaining the final concentrations of 0.5, 1.0, 1.5, 2.0, 4.0 and 6.0 mg mL -1 according to Di Piero and Garda (2008).The absolute control contains only the PDA medium, and the relative control contains PDA and acetic acid in the same volume as the gel chitosan (Bautista-Baños et al., 2016).
The chitosan gel was sterilized for 15 min at 120°C and transferred to Petri dishes until complete growth, and then, the mycelia of S. rolfsii (0.5 cm) was added in the center of each Petri dish and maintained in camera BOD at 28°C.The diameter of the fungus was determined every 24 h until the control treatment reached the plaque boards.The fungal growth was observed in subsequent periods (7, 14, 21, 28 and 35 days) in the different treatments (0.5, 1.0, 1.5, 2.0, 4.0 and 6.0 mg mL -1 ), and two control treatments were applied: (a) absolute control (PDA) and (b) relative control (PDA + Acetic acid), with six replicates.
The data were submitted to statistical calculations using the software program SAS Institute, Learning Edition 9.1, Cary, North Carolina, USA.Analyses of variance and means were compared by the Tukey test (P≤0.05),and when necessary, the regression analyses were processed to evaluate the effectiveness of the chitosan concentration on fungal growth as a function of the subsequent periods (Stamford et al., 2017).

Greenhouse experiment in soil pots
The mixed biofertilizer was produced from phosphate and potassic rock biofertilizers mixed with earthworm compost as organic matter (OM) in a PKB:OM ratio of 1:3 (v/v) following Stamford et al. (2007).The P and K analysis for rock biofertilizer showed the following results: pH = 3.8, available P = 60 g kg -1 and available K = 10 g kg -1 (Silva et al., 2009).
The earthworm compost was enriched in N by inoculation with the free living diazotrophic bacteria B. indica (NFB 1001) selected in the Nucleus of Nitrogen Fixation at the University Federal Rural of Pernambuco (UFRPE).The selected diazotrophic bacteria were cultured in LG liquid media (50 mL) in 125-mL Erlenmeyer flasks and shaken (180 rpm) for 96 h at ± 28°C, and 100 mL was applied per tray (6 L) according to Lima et al. (2010).Analysis of the earthworm compound showed the following: pH = 7.85; organic carbon = 120.7 g kg -1 ; total N = 8.6 g kg -1 ; total sulfur = 2.9 g kg -1 ; and total P = 11.2 g kg -1 .The mixed organic biofertilizer analysis showed the following: pH = 6.2; organic carbon = 100 g kg -1 ; total N = 20 g kg -1 ; total sulfur = 3 g kg -1 ; available P = 13 g kg -1 ; and available K = 15 g kg -1 (Silva et al., 2009).
To produce the bioprotector, the organic biofertilizer enriched in N was inoculated with a mycelia biomass of the Mucorales fungi C. elegans (UCP 542) that contained chitosan and chitin in the cellular wall (Berger et al., 2016).The fungus was grown for seven days at 28°C in Petri dishes with liquid PD medium, producing a final concentration of 10 6 spores mL -1 , and the spore suspension was shaken at 150 rpm.
The greenhouse experiment was carried out at the University Federal Rural of Pernambuco from 03 June to 23 July 2016, using an Oxisol soil (USDA, 2014).The soil samples were collected at 0 to 20 cm deep from the rain forest region, Pernambuco, Northeast Brazil, with coordinates 7° 53′ 49″ S, 35° 10′ 48″ W. The soil was air dried sieved (5 cm), mixed and kept in pots (8 dm 3 ).Chemical analysis of the soil showed the following: pH (H2O) = 5.4 and exchangeable cations (cmolc dm -3 ): K + = 0.08; Ca 2+ = 1.40; and Mg 2+ = 0.55.Soil analyses were processed using an absorption spectrophotometer and atomic emission (Perkin Elmer 3110), and total N was determined by the Kjeldhal methodology using a N auto analyzer (Kjeltec 1030).
The experiment was realized with sterilized and non-sterilized soil to evaluate the effectiveness of the bioprotector inoculated with the Mucorales fungi C. elegans and the action of crustaceous chitosan (foliar application) against the pathogen S. rolfsii on cowpea.The soil sterilization was processed three times for 60 min at 121°C in intervals of 24 h.
During the experiment, the photoperiod remained close to 12 h of dark and 12 h of light, the temperature oscillated between 28 to 36°C, and the relative humidity was 60 to 80%.Seven days after germination, the crustaceous chitosan was sprayed on the first pairs of completely developed leaves (4 mL plant -1 ).The plants were inoculated with the pathogen S. rolfsii by addition of a conidial suspension (10 6 mL -1 ).
The index of disease severity was evaluated at 50 days after the planting date by a transversal cut in the stem of the plants to observe the occurrence of symptoms in the vascular system.The index of disease severity was processed as described earlier: (1) No visual symptoms of the disease in plants and in soil; (2) Small symptoms (necrosis in the shoot of plants) at the point of inoculation; (3) Large symptoms (large visual lesions in the shoot of plants) and fungal growth, plants showed debilitation, and the soil has fungal growth; (4) Plants present visual symptoms of wilt in the hypocotyls, showing fungal growth and the presence of scleroses in the plant and soil, and some plants are dead; (5) Many plants are dead or showed large symptoms of wilt, showing fungal growth in the plant and soil.
The activities of enzymes (catalase and peroxidase) were evaluated in cowpea at 8 and 16 days after pathogen inoculation.The enzyme extract was prepared with 1.0 g of macerated leaf tissue using liquid nitrogen, 4 mL of phosphate buffer solution and 50 mg of peroxidase.This concentrate was filtered and centrifuged (10 min at 4°C, 10000 g).The supernatant was stored at -80°C and used to evaluate the activity of catalase and peroxidase via the soluble protein content according to Andrade et al. (2013).The soluble protein was determined by colorimetric analysis (Bradford, de Souza et al. 10551976).
The peroxidase activity was estimated with the evaluation of Δ absorbance provided through the oxidation of guaiacol (C3H8O2) in the presence of hydrogen peroxide (Fatibello-Filho and Vieira, 2002).The catalase activity was analyzed according to Beers and Sizer (1952).
The statistical calculations were performed using the software program SAS Learning Edition 9.1.Analyses of variance and means were compared by the Scott-Knott test (P≤0.05)(SAS, 2011).

Mycelia growth of S. rolfsii: In vitro assay
In the mycelia growth of S. rolfsii, a significant difference was observed when chitosan was used at concentration of 6.0 mg mL -1 , and the application of chitosan showed fungicidal ability when applied in lower concentrations (2 and 4 mg mL -1 ) with a total effect on S. rolfsii growth (Figure 1).
The chitosan concentration demonstrated a significant fungistatic effect on the growth of S. rolfsii, and an effect of the acetic acid on the fungi growth was not observed.
The control treatment affected the fungal growth after three days of incubation until the control treatment reached the plaque boards (Figure 1G).The relative control was not different from the absolute control, and the fungal growth was affected within four days of incubation (Figure 1G).The crustaceous chitosan showed fungistatic and fungicidal effects and promoted morphological changes of S. rolfsii during the growth period.

Bio-protector production
The strain of B. indica (NFB 1001 and 1003) used to produce the bioprotector was completely characterized by the Korean group Macrogen Incorporation(Geumcheon-gu Mycelia growth of Sclerotium rolfsii with application of crustaceous chitosan at different concentrations (0.5, 1.0, 1.5, 2.0, 4.0, and 6.0 mg mL -1 ), including absolute and relative controls at various growth times (0, 7, 14, 21, 28 and 35 days) Seoul), and the gene sequences compared with other diazotrophic bacteria are shown in Table 1.The index of disease severity for S. rolfsii (%) in cowpea collected 50 days after pathogen germination is shown in Table 2.The chitosan applied in cowpea culture increased the defense against S. rolfsii.Plants with less damage were The results for the index of disease severity were higher in sterilized soil than in non-sterilized soil.The treatments with crustaceous chitosan at 6.0 and 4.0 mg mL -1 in sterilized and non-sterilized soil, respectively, resulted in a lower index of disease severity in cowpea plants.In nonsterilized soil, crustaceous chitosan at 4.0 mg mL -1 showed a lower index of disease severity that was significantly different in the cowpea plants.A low index of disease severity was obtained when the bioprotector with an increased fertilizer concentration was applied, especially at the highest rate (150% RR), which presented an index of disease severity of 1.5% in sterilized soil and 1.8% in non-sterilized soil.
The bioprotector applied at the different concentrations produced higher soluble protein content compared with the treatments of earthworm compost without chitosan, earthworm compost with chitosan (rates 2.0 and 6.0 mg mL -1 ) and mixed biofertilizer with chitosan (Table 3).
An interaction effect was observed when the mixed biofertilizer was applied at higher rates and increased the protein content.However, the treatments using mixed biofertilizer with pathogen and the control treatment presented the higher protein content in sterilized soil.
In the second period, an effect of the control treatment with lower protein content (P<0.05%) was observed compared with the other treatments (Table 3).In non-sterilized soil, the mixed biofertilizer showed a higher soluble protein content, and the bioprotector, independent of the concentration, showed a lower protein content (P<0.05).In the sterilized soil, the treatment with earthworm compost and chitosan (4.0 and 6.0 mg mL -1 ) with pathogen inoculation produced higher protein content.

Peroxidase activity
Peroxidase activity in cowpea leaves collected eight days after inoculation with S. rolfsii using sterilized and non-sterilized soil is shown in Table 4.The peroxidase activity was activated in the leaves of cowpea plants when crustaceous chitosan was applied at higher levels (bioprotector + chitosan at 6 mg mL -1 ).Consequently, the plants that received this treatment showed less damage than the control treatment.
In non-sterilized soil, the plants inoculated with S. rolfsii that received treatments with bioprotector + chitosan (4 mg mL -1 ) and bioprotector + chitosan (6 mg mL -1 ) showed a higher peroxidase activity compared with the plants in the absence of this pathogen.The application of these treatments also resulted in the lowest index of disease severity of S. rolfsii wilt in the cowpea plants.Consequently, the relationship between increases in peroxidase activity and increases in plant protection in the presence of a biotic stress is confirmed.
The results of peroxidase activity in the cowpea leaves collected 14 days after inoculation with S. rolfsii are shown in Table 4.The enzyme peroxidase showed low activity in the plants with the bioprotector + chitosan (6.0 mg mL -1 ) and bioprotector + chitosan (4.0 mg mL -1 ) treatments with sterilized and non-sterilized soil, respectively.In sterilized soil, it was observed that the treatments induced higher peroxidase activity, especially when compared with the control treatment that used soil without pathogen addition.

Catalase activity
In the first period, independent of the sterilized and non-sterilized soil, the treatment of earthworm compost with the treatment with the bioprotector in a higher rate (150% RR) presented higher catalase activity.
The catalase activity in cowpea leaves collected eight days after inoculation with S. rolfsii is presented in received the bioprotector + chitosan (6.0 mg mL -1 ) showed higher catalase activity.Table 3. Soluble protein in leaves collected at 14 and 28 days after pathogen inoculation (DAPI) on cowpea in an Oxisol of the Brazilian rain forest region in sterilized and non-sterilized soil submitted to different fertilization treatments.This result probably contributed to the lowest index of disease severity for S. rolfsii wilt in the cowpea plants.

Treatment
The same relationship between the increase in enzyme activity and the reduction in the disease severity index was already discussed with respect to the greater peroxidase activity of the bioprotector with chitosan (4.0 and 6.0 mg mL -1 ) treatments observed in the nonsterilized soil.
In the non-sterilized soil, the low index of disease severity in the plants inoculated with S. rolfsii treated with chitosan at higher concentration showed a reduction in the catalase activity after the pathogen application.The sterilized soil show a significant decrease in the catalase activity when compared with the results in leaves collected eight days after inoculation with the pathogen.
The reduction in catalase activity after the pathogen inoculation (Table 5) compare the results in Table 4 may occur due to the reduction in H 2 O 2 in the plants, and the activation of the antioxidant enzyme may be unnecessary.The low activity of catalase is possible due Table 4. Peroxidase activity in leaves of cowpea collected at 14 and 28 days after pathogen inoculation (DAPI) in an Oxisol of the Brazilian rain forest region, using sterilized and non-sterilized soil submitted to different fertilization treatments.to the low amount of ROS in the sites where the enzymes act, which are sites of the peroxidase action.

Effectiveness of chitosan in vitro
The effectiveness of chitosan on Botrytis cinerea was reported by Camili et al. (2007), who described the in vitro effects of chitosan concentrations equivalent to 0.5, 1.0, 1.5 and 2.0% over a period of five days at 22°C.Similar results were reported by Berger et al. (2016) with crustaceous chitosan, and the authors observed a fungistatic effect in the control of Fusarium oxysporum f.sp.tracheiphilum.
The dependence of the effect of chitosan on the concentration was also reported by Freddo et al. (2014) in a study with Rhizoctonia solani to evaluate the fungistatic effect of chitosan applied in various concentrations (0.0, 0.25, 0.5, 1.0 and 2.0%).Liu et al. (2006) observed a fungicidal effect of chitosan inhibiting the mycelia growth and spore germination of Botrytis cinerea and Penicillium expansum fungi.
Many authors suggested that the antimicrobial activity of chitosan is due to the amino group in a polycationic form in the presence of low pH, as generally occurs in chitosan solutions.In these conditions, the cationic structure may interact with the negative charges of the anionic groups of the cellular membrane of the microorganisms and change the permeability, promoting the decrease in intracellular components (Di Piero and Garda, 2008).Prapagdee et al. (2007) agree that the effect of chitosan is a fungistatic effect, because this biopolymer is the principal active compound that can promote structural and morphological modifications and can disorganize the molecules of the fungi.

Gene sequence of the diazotrophic bacteria
Most of the genes involved in the nitrogen fixation process by the diazotrophic bacteria (strain NFB 1001) are distributed into two genomes (10 kb and 51 kb) regarding the gene nif that responds to the codification of cysteine desulfurase (Tamas et al., 2010).The strain NFB1001 showed 99% similarity and covertures of 73% with the B. indica species.It is found to have lower similarity with the others diazotrophic bacteria deposited in "GenBank" compared with the diazotrophic isolates used in this study.The sequence of the gene nif may involve the best information about the diazotrophic bacteria because this gene sequence can confirm 100% of the diazotrophic isolates.However, the gene nif has several limitations to its use in phylogenetic analyses and depends on the microbial group (Gaby and Buckley, 2012).

Effectiveness of the bio-protector and chitosan in foliar application
Several studies in greenhouse conditions have shown that chitosan is a polymer that could potentially be used to control plant disease (El Hadrami et al., 2010;Soleimani and Kirk, 2012).When chitosan is applied as a root or seed dressing and in foliar spray, such as in this experiment, the biopolymer can hinder pathogen growth in the plant host tissue.Lowe et al. (2012) observed that chitosan, applied as a foliar spray, reduced the disease symptom severity in strawberry plants infected by Bacillus subtilis.The biopolymer induces a number of defense reactions, including structural barriers and different biochemical activities during the plant-pathogen interaction.In response to chitosan application, the host plants showed cellular lignifications and fungal toxin accumulation at sites of attempted pathogen penetration.The chitosan created a barrier that impeded the flux of nutrients between the host and the pathogen, and these results are in accordance with Bautista-Banõs et al. (2006).
In a study to evaluate the effectiveness of crustaceous chitosan, Berger et al. (2016) reported no effect of the biopolymer on the growth of F. oxysporum f.sp.tracheiphilum when used at low concentrations, and a great effect in high concentration (4 and 6 mg mL -1 ).Most likely, the chitosan contained in the bioprotector inoculated with C. elegans that is present in fungi as chitosan in the cellular walls may act against pathogens.In accordance with Stamford et al. (2017), this biopolymer induces resistance against pathogens.Di Piero and Garda (2008) demonstrated the effectiveness of crustaceous chitosan in the control of Colletotrichum lindemuthianum on Phaseolus vulgaris, with an increase in the activity of the enzyme β-1,3-glucanase that directly affects the glucose present in the cellular walls, inhibiting the fungal growth.According to Thakkar and Sarafi (2015), chitosan affects the biological control of pathogens because the biopolymer increases the plant resistance.
It is very important to observe that when plants grow in sterilized soil show a low response to the chitosan application against phytopathogenic microorganisms and a non-significant change compared with the control treatment.This behavior may occur in relation to the alterations verified in the soil biota, probably due to the soil autoclaving process.Soil exposed to high temperatures during the autoclave process has reduced native microorganisms, and the competition against S. rolfsii increases the pathogen growth and directly contributes to an increase in the index of disease severity in accord with (Stamford et al., 2017).

Enzymatic activity
The results of enzymatic activity suggested that the increase in peroxidase activity provides protection against S. rolfsii.On the other hand, chitosan can induce peroxidase activity in plants in the absence of a pathogen.The same treatment with bioprotector + chitosan (6 mg mL -1 ) also resulted in greater peroxidase activity in cowpea plants in the absence of a pathogen.Falcón-Rodríguez et al. (2009) demonstrated the increase in peroxidase activity in leaves and roots of tobacco plants treated with chitosan in the absence of pathogens.These authors suggested that chitosan induces defensive enzyme activity and reported that the plants accumulate secondary metabolites and form barriers to enhance plant resistance against pathogens.
The peroxidase activity may directly influence the control of pathogens because it liberates nonspecific compounds that increase plant defenses (Van Loon et al., 2006).In the same way, chitosan has been characterized as a signal in the plant response and may minimize the plant response against pathogens (Mejía-Teniente et al., 2013).In addition, the performance of peroxidase activity in plant resistance against pathogen attacks is based on hydrolytic activities in the cellular walls, where direct or indirect effects may increase antifungal activities (Stangarlin et al., 2011).
No significant difference was observed in the peroxidase activity in plants inoculated with the pathogen.However, the plants that received the bioprotector + chitosan (2 mg mL -1 ) shows a decrease in the peroxidase activity, and it may be conclude that the crustaceous chitosan at this concentration (2 mg mL -1 ) does not induces peroxidase activity in cowpea plants.
The chitosan can induce the antioxidant system and increase the peroxidase activity and increases the plant protection against some stressors, as described by Ortega-Ortiz et al. (2007).These authors suggested that the effects of chitosan on the disease control and quality maintenance in peach fruit may be associated with their antioxidant properties and the elicitation of defense responses.Mazaro et al. (2012) reported that chitosan treatment reduced Mycosphaerella dendropoma in strawberry plants and activated peroxidase activity.The authors suggested that the alteration in peroxidase activity may be reflected by a metabolic response of the plant that leads to lignin formation.
The antioxidant enzyme peroxidase is also responsible for the control of pathogenic microorganisms because it increases the production of ROS and is a signal of the plants to biotic stress promoted by a pathogen (Sharma et al., 2012).Furthermore, avoiding cellular damage is important to control the equilibrium between ROS formation and the detoxification of the antioxidant enzymes such as peroxidase and catalase.
In accordance with Dousseau et al. (2016), peroxidase and catalase enzymes can increase the oxidation of phenol groups as a precursor of lignin synthesis and increase the resistance of the cellular wall for pathogenic entrance in infected plants.These authors reported the effect of different chitosan concentrations (2.5, 5.0 and 10.0 g L -1 ) on the catalase activity in jaborandi (Piper mollicomum), and the best results were obtained using the concentration of 5 g L -1 . The results of catalase activity suggest that the chitosan concentration reflects an increase in the enzyme activity and that the higher enzyme activity in cowpea directly influences the plant age.Ortega-Ortiz et al. (2007) also find the same relationship in the peroxidase and catalase in tomato fruit during different growth states of the fruit.The authors agree that the resistance inductor chitosan de Souza et al. 1061 has different effects according to the stage of development of the fruit.

Conclusions
The in vitro test demonstrates that the crustaceous chitosan promotes fungicide and fungistatic effects on the mycelia growth of S. rolfsii.The experiment in soil pots showed that the bio-protector and crustaceous chitosan applied in different concentrations reduces the index of disease severity of S. rolfsii wilt on cowpea and induces mechanisms of defense in cowpea, increasing soluble protein, peroxidase, and catalase activity.
the same letter are not different in treatments according to the Scott-Knott test (P≤0.05).
the same letter are not different in treatments according to the Scott-Knott test (P≤0.05)Table5.Catalase activity in leaves of cowpea collected at 14 and 28 days after pathogen inoculation (DAPI) in an Oxisol of the Brazilian rain forest region using sterilized and non-sterilized soil submitted to different fertilization treatments.the same letter are not different in treatments according to the Scott-Knott test (P≤0.05).

Table 1 .
Characterization and rRNA 16S gene sequence of the diazotrophic bacteria isolated from Brazilian soils, compared with other diazotrophic bacteria deposited in GenBank (NCBI).

Table 2 .
Index of disease severity for Sclerotium rolfsii on cowpea in Oxisol soil in the Brazilian rain forest region, evaluated at 50 days after seed germination in sterilized and non-sterilized soil submitted to different fertilization treatments.