ALLEVIATING STRESS OF SCLERTIUM ROLFSII ON GROWTH OF CHICKPEA VAR. BHAKKAR-2011 BY TRICHODERMA HARZIANUM AND T. VIRIDE
A. Javaid*, A. Ali, A. Shoaib and I. H. Khan
Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan.
*Corresponding Author’s email: firstname.lastname@example.org, email@example.com
Chickpea (Cicer arietinum L.) is attacked by Sclerotium rolfsii at the seedling stage and the resulting collar rot disease significantly reduces the survival percentage of the seedlings and ultimately yield of the crop. In order to reduce environmental pollution caused by the use of synthetic fungicides, this study was carried out to use two biocontrol agents, namely Trichoderma harzianum and T. viride, against S. rolfsii, and to investigate their effect on plant growth, yield and physiology of chickpea var. Bhakkar-2011. S. rolfsii inoculation reduced dry weight of shoot, root and grains by 21.4%, 36.5% and 49%, respectively, over negative control. T. harzianum and T. viride increased shoot dry weight by 120% and 362%, root dry weight by 132% and 138%, and grain yield by 1109% and 572%, respectively, over positive control (S. rolfsii inoculated only). The effects of the pathogen and the two biocontrol agents were also studied on chlorophyll, carotenoid and phenolic contents as well as on activities of antioxidant enzymes viz. peroxidase (POX), phenylalanine ammonia lyase (PAL) and catalase (CAT). S. rolfsii inoculation suppressed chlorophyll and carotenoid contents while both the Trichoderma spp. increased these parameters many folds. Phenolic content and activities of POX, PAL and CAT were generally increased due to S. rolfsii but became normal due to application of Trichoderma spp. This study concludes that T. harzianum and T. viride are the potential biocontrol agents for control of collar rot of chickpea var. Bhakkar-2011.
Keywords: Biocontrol, chickpea, Sclerotium rolfsii, Trichoderma harzianum, Trichoderma viride.
Published online March 31, 2021
Sclerotium rolfsii Sacc. is a serious pathogen in tropical and sub-tropical areas with a wide host range of about 500 species of plants (Jacob et al., 2018). Being soil-borne in nature, it is responsible for collar rot, an emerging plant disease that may incite 65–90% mortality of chickpea seedlings. High soil moisture in combination with 28-30°C temperature are the favorable environmental conditions for its growth and infection (Pravi et al., 2015; Rajani et al., 2019). At the initial stages of infection, it may result in stem decay followed by wilting and plant death. At advanced infectious stages, the stem near the soil line bears white mycelial growth and tan to brown sclerotial bodies (Queiroz et al., 2017; Zheng et al., 2020). The pathogen can survive in plant tissues or in plant debris that upon conducive conditions attack the chickpea collar region (Tarafdar et al., 2018).
Management of S. rolfsiiis is somewhat challenging due to its prolific growth, wide host range, and having the capability to form abundant sclerotia that may remain in the soil up to 7 years under harsh conditions (Rodriguez-Kabana et al., 1980; Bholanath and Papiya, 2017). Many efforts have been employed to control this devastating pathogen but met with limited success due to lack of sufficient information (Lal et al., 2015). Different fungicides are in practice but they are not economical in the long run because they damage the environment, pollute the atmosphere and their repeated use also leaves detrimental effects on beneficial microorganisms (Kumar et al., 2018). Of the various methods used to manage S. rolfsii, the use of biocontrol agents gave promising results with little or no hazardous effects on the environment (Rajani et al., 2019; Javaid et al., 2020; Sharf et al., 2021). In current years, biocontrol of soil-borne diseases by microbial antagonists has been widely reported (Rabinal and Bhat, 2020; Shoaib et al., 2020). Trichoderma spp. are widespread saprophytic, soil-inhabiting filamentous fungi that have the ability to antagonize various pathogenic fungi resulting in reduced disease incidence (Chen et al., 2016; Khan et al., 2021). Mycoparasitism, cell-wall degrading enzymes and antifungal compounds production, antibiosis and competition for space and nutrients are the possible mechanisms of actions of these fungi against the fungal pathogens (Divya and Sadasivan, 2016; El-Sobky et al., 2019; Khan and Javaid, 2020). The present study was undertaken to assess the beneficial effects of the application of two Trichoderma species on growth and physiology of chickpea variety Bhakkar-2011 growing in S. rolfsii contaminated soil.
MATERIALS AND METHODS
Pot trial: The effectiveness of the two Trichoderma species against S. rolfsii on chickpea var. Bhakkar-2011 was scrutinized under soil treatment conditions following methods described by Javaid et al. (2020). Inoculations of the Trichoderma species and S. rolfsii were conducted according to the following combinations: T1: negative control; T2: positive control [S. rolfsii (SR)]; T3: T. harzianum (TH) + SR and T4= T. Viride (TV) + SR. There were five replications per treatment. T. harzianum, T. viride and S. rolfsii were separately propagated on pearl millet seeds in conical flasks, and the grain cultures were used as inocula for the respective treatments. Soil was fumigated with wet balls of cotton wool dipped in formalin, and was filled in the pots (5 kg/pot). For T2–T4, sterilized soil was inoculated with S. rolfsii (50 g/pot) propagated on the pearl millet grains, and kept for 7 days for the pathogen establishment. For T1, the pots were mixed with the same quantity of boiled grains without any inoculum. However, in the case of T3 and T4, the pathogen inoculated pots were also inoculated (50 g/pot) with TH and TV, respectively and kept for another 7 days. Chickpea seeds were surface sterilized with 1% NaOCl solution and were sown at 10 seeds/pot). All the pots were watered regularly, arranged randomly and kept under the natural environmental conditions. The plants were harvested at maturity and, shoots and roots were separated. Roots were washed gently over a sieve using tap water. Data regarding root and shoot lengths, as well as dry weights of shoots, roots, pods and grains were recorded.
Estimation of chlorophyll and carotenoid contents: Leaf sample was grounded in 80% ethanol and the homogenized sample was centrifuged at 10,000 rpm for 5 min, while the supernatant was assessedfor chlorophyll a, chlorophyll b, and carotenoids by recording absorbance at 645 nm, 663 nm, and 270 nm, respectively. Total chlorophyll content and carotenoids were calculated using the formula specified by Lichtenthaler and Wellburn (1983).
Estimation of phenolic content: Total phenolic content of ethanolic leaf extract was estimated using Folin-Ciocalteatou reagent and taking absorbance of the mixture at 765 nm (Singleton and Rossi, 1965).
Estimation of antioxidant enzymes: CAT activity of the reaction mixture (leaf extract + 75 mM phosphate buffer + 112 mM H2O2) was examined as change in absorbance as a result of H2O2 consumption at 240 nm (Havir and McHale, 1987).
For POX assay, a reaction mixture was prepared by adding 25 mM phosphate buffer, 20 mM pyrogallol and 20 mM hydrogen peroxide in the leaf extract. The amount of purpurogallin formed was determined by measuring the absorbance at 420 nm (Kar and Mishra, 1976).
PAL activity was analyzed in the reaction mixture consisted of leaf extract, 0.1 M sodium borate buffer and 12 mM phenylalanine. A change in absorbance was recorded at 270 nm spectrophotometrically (Cochrane et al., 2004).
Statistical analysis: One-way ANOVA was applied to analyze the data regarding root and shoot growth, grain yield, chlorophyll, carotenoid and phenolic contents, and activities of POX, PAL and CAT, followed by the application of LSD test to determine significant differences among the treatments mean at P≤0.05 using Statistix 8.1.
RESULTS AND DISCUSSION
Effect of S. rolfsii and Trichoderma spp. on plant growth and yield: Application of S. rolfsii (T2) reduced all the growth and yield related parameters when compared with uninoculated negative control treatment (T1). Addition of the pathogen reduced shoot length and biomass by 17.3% and 21.4%; root length and biomass by 26.10% and 36.5%, and dry weights of pods and grains by 39% and 49%, respectively, over T1 (Fig. 1 and 2). S. rolfsii is a highly problematic soil-borne fungal pathogen and is also responsible for causing similar reductions in growth and yield of many other plant species including chickpea (Khan et al., 2020), mungbean (Sun et al., 2020) and tomato (Sahu et al., 2019).
Fig. 1: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on shoot and root growth of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
Fig. 2: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on pod and grain yield of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
Both the Trichoderma species improved plant growth and yield under the stress of S. rolfsii. Boost in growth and yield due to the inoculation of Trichoderma species was generally significantly higher than T1 and T2. Earlier studies have shown that different species of Trichoderma have the potential to alleviate the stress of various pathogens and improve crop growth and yield (Akramiet al., 2011; Liu et al., 2020). Physical as well as chemical mechanisms are involved in the control of fungal pathogens and an increase in plant growth by application of Trichoderma spp. Different substances are released by Trichoderma spp. which provoke resistance in the host against the disease-causing agents (Gary et al., 2004). Some species of Trichoderma also act as antagonists against pathogenic fungal species (Doley and Jite, 2012; Al-Ani and Mohammed, 2020). Recently, it has been reported that Trichoderma species suppress the growth of M. phaseolina through its DNA disintegration (Khan and Javaid, 2020; Khan et al., 2021).
T. harzianum increased shoot length by 98% and 139%, shoot dry weight by 82% and 132%, root length by 29% and 74%, root dry weight by 40% and 120%, pod weight by 280% and 533% and grain yield by 504% and 1109% over negative and positive control treatments, respectively. T. harzianum is known for its biocontrol activity against a number of phytopathogenic fungi. It is known for its biocontrol activity against Sclerotinia sclerotiorum in soybean as reported by Zhang et al. (2016), Macrophomina phaseolina in mungbean (Javaid et al., 2017), Fusarium oxysporum f. sp. cepae in onion (Akhtar and Javaid, 2018) and Fusarium solani in olive trees (Amira et al., 2017). Earlier, Rekha et al. (2012) reported that metabolites of T. harzianum limited the formation of zoospore and germ tube, and the growth of mycelium of S. rolfsii. Youssef et al. (2016) reported that the protection provided by T. harzianum against Rhizoctonia solani was related to increase in activities of guaiacol peroxidase, ascorbate peroxidase, catalase and superoxide dismutase. Recently, Bader et al. (2020) demonstrated that native Argentina strains of T. harzianum produce auxin indole 3-acetic acid, solubilize phosphate, enhance growth and control tomato wilt caused by F. oxysporum. The aspartic protease P6281 produced by T. harzianum plays a vital task in mycoparasitism on plant pathogenic fungal species (Deng et al., 2018).
Fig. 3: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on leaf chlorophyll and carotenoid contents of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
Fig. 4: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on phenolic content of leaf and root of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
Fig. 5: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on peroxidase activity (POX) of leaf and root of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
Fig. 6: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on phenylalanine ammonia lyase activity (PAL) of leaf and root of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
Fig. 7: Effect of Sclerotium rolfsii (SR), Trichoderma harzianum and T. viride on catalase activity (CAT) of leaf and root of chickpea var. Bhakkar-2011. Vertical bars show standard errors of means of five replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD Test.
T. viride application was also proved highly beneficial resulting in an increase of 94% and 135% in shoot length, 87% and 138% in shoot dry weight, 104% and 176% in root length, 194% and 362% in root dry weight, 260% and 500% in pod weight and 236% and 572% in grain yield over T1 and T2, respectively. Earlier reports indicated that T. viride has the potential to control in vitro development of other pathogenic fungi namely M. phaseolina and F. oxysporum (Javaid et al., 2014, 2018). It has been found effective in controlling brown blotch disease of cowpea through the production of an antifungal compound viridian (Bankole and Adebanjo, 1996). It is also known to control seed-associated fungi, namely Fusarium moniliforme and Aspergillus flavus through production of lipolytic, pectinolytic proteolytic and cellulolytic enzymes (Calistru et al., 1997). It was also proved as a highly effective biocontrol agent against root pathogens of soybean namely Pythium arrhenomanes and F. oxysporum f. sp. adzuki resulting in improved crop growth (John et al., 2010).
Effect of S. rolfsii and Trichoderma spp. on plant physiology: Photosynthetic pigments were significantly decreased under pathogenic stress (T2) as compared to un-inoculated control (T1).However, either of the Trichoderma species proved very effective in tremendously improving the said attributes up to 5-folds as compared to T2. Reduction in the photosynthetic pigments in T2 indicated that biotic stress might cause irregularities in photosynthesis and respiration rates, which generally causes an intensified level of reactive oxygen species (ROS). Hence reduced chlorophyll content is a typical symptom of stressed plants incurred by the pathogen (Nafisa et al., 2020). Likewise, total phenolic content was increased and activities of PAL, CAT and POX were significantly higher in T2 with respect to T1, which is in agreement to the earlier studies (Hossain et al., 2016; Shoaib et al., 2018; Nafisa et al., 2020). Higher enzyme activities may be correlated with higher ROS accumulation in the stressed cells as enzymes contribute to lowering the ROS levels. Furthermore, higher CAT and POX may show their higher consumption as a result of oxidative stress. However, it seems that the chickpea plant might not be able to handle stress posed by the pathogen, which resulted in disease in plants. Biocontrol agent’s inoculations seem effective as either species of Trichoderma has been known to proliferate faster, colonizes root surface and secrete cell wall hydrolyzing enzymes along with bioactive compounds, which likely to limit pathogen growth as well enhanced plant resistance. Besides, Sclerotium spp. are known to secrete some lectins, which can stimulate Trichoderma to coil around pathogen hyphae (Srivastava et al., 2015). Therefore, treatments provided with biocontrol organisms in the form of T. viride and T. harzianum normalized the effects on chickpea plants, which would have to face in case of pathogen attack.
Conclusion: This study clearly indicates that T. harzianum and T. viride have pronounced the potential to increase crop growth and yield of chickpea var. Bakhar-2011 many folds under the biotic stress of S. rolfsii by regulating plant physiology.
Author’s contribution: Amna Ali carried out experimental work. Arshad Javaid supervised the whole work, wrote a part of the paper, and did the statistical analysis. Amna Shoaib did physiological studies. Iqra Haider Khan contributed to the writing of the manuscript.
Conflict of interest: The authors declare no conflict of interest.
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