Glomus fasciculatum in defense responses to stop the rot of Arachis hypogaea L.

Glomus fasciculatum en las respuestas de defensa para detener la putrefacción de Arachis hypogaea L.

Khirood Doley^*, Mayura Dudhane, Mahesh Borde

¹ Department of Botany, Mycology Laboratory, University of Pune, Maharashtra, India. * Author for correspondence.

Received: September, 2014.
Approved: June, 2015.

Abstract

Arbuscular mycorrhizal (AM) fungi can play the potential role of bio-control agent, which will enable a putative perspective in bio-protection and intensive organic agricultural practices since chemical pesticides are associated with several health hazards. The objective of this pot culture research was to study the roles played by AM fungi (Glomus fasciculatum) in the biocontrol of pathogen Sclerotium rolfsii, which is a causal agent of stem-rot in groundnut plant. The experimental design was randomized complete block with four treatments and three replicates per treatment; data were analyzed with ANOVA and treatments means were compared using Tukey test (p≤0.05). Groundnut plants were treated with AM S. rolfsii or both after 30, 60 and 90 d of planting. The soil based AM fungi were inoculated before pathogen inoculation. Results showed that S. rolfsii caused stem-rot disease in groundnut plants as expected, but the AM fungi inoculation significantly enhanced host plant endurance by decreasing stem-rot severity, and increased growth variables of inoculated plants. Besides, the effect of AM fungi was noticeable in root colonization and mycorrhizal dependency percentage, and there was a significant increase in content of total chlorophyll, protein, total phenols and polyphenol activities in mycorrhizal groundnut plants inoculated with pathogen. The antioxidant related response such as peroxidase was higher, reflecting a possible defense resistance mechanism induced upon by the AM fungi colonization by reducing the harmful effects of reactive oxygen species produced during the growth of groundnut plants due to S. rolfsii.

Keywords: AM fungi, anti-oxidant enzymes, biocontrol, G. fasciculatum, groundnut, S. rolfsii.

Resumen

Los hongos micorrízicos arbusculares (MA) pueden tener la función potencial de agente de biocontrol, lo cual permitirá tener una perspectiva supuesta sobre la bioprotección y las prácticas agrícolas orgánicas intensivas, dado que los pesticidas químicos están asociados con daños serios a la salud. El objetivo de esta investigación fue el cultivo en maceta para estudiar la función que tiene el hongo MA (Glomus fasciculatum) en el biocontrol del patógeno Sclerotium rolfsii, que es un agente causal de la pudrición del tallo en la planta de cacahuate. El diseño experimental fue bloques completos al azar con cuatro tratamientos y tres réplicas por tratamiento; los datos se analizaron con ANDEVA y las medias de los tratamientos se compararon con la prueba de Tukey (p≤0.05). Las plantas de cacahuate se trataron con hongo MA S. rolfsii o ambos después de 30, 60 y 90 d de plantar. Los hongos MA del suelo fueron inoculados antes de la inoculación con el patógeno. Los resultados mostraron que S. rolfsii causó enfermedad de pudrición del tallo en plantas de cacahuate, como se esperaba, pero la inoculación con hongos MA mejoró significativamente la resistencia de la planta al disminuir la severidad de la pudrición del tallo y aumentar las variables de crecimiento de las plantas inoculadas. Además, el efecto de los hongos MA fue notable en la colonización de las raíces y en el porcentaje de dependencia micorrízica, y hubo un aumento significativo en el contenido de clorofila total, proteína, fenoles totales y actividades de polifenoles en las plantas de cacahuate micorrízicas inoculadas con el patógeno. La respuesta relacionada con antioxidantes como la peroxidasa fue mayor, reflejando un posible mecanismo de resistencia de defensa inducido por la colonización del hongo MA al reducir los efectos dañinos de las especies reactivas de oxígeno durante el crecimiento de las plantas de cacahuate, debido al S. rolfsii.

INTRODUCTION

The peanut or groundnut (Arachis hypogaea L.) is an important oilseed crop and food legume grown on about 20 million ha in warm tropical or subtropical areas throughout the world (Wyne and Beute, 1991). Among producers of groundnut India ranks second, but the rate of production does not keep pace with the domestic demand, despite the increase in production of groundnut; thus, India imported 5 million Mg soybean oilcake meals 5 during 2003-2007 (Birthal et al., 2010). The main cause of this situation is a severe yield decrease in groundnut growth and production caused by bacterial, viral or fungal pathogens (Roy and Shiyani, 2000). Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii) (Curzi), a soil-borne fungal pathogen, shows destructive ability in groundnut plants and it is the causal agent of stem-rot (Narain and Kar, 1990). In crop debris S. rolfsii survives in the form of sclerotia and is found in the tropics, subtropics and other warm temperate regions (Punja, 1985).

Several chemicals compounds are available against soil-borne plant pathogens, but the employment of biological control agents is inevitable due to environment protection (Naseby et al., 2000). Arbuscular mycorrhizal (AM) fungi belonging to phylum Glomeromycota, which inhabits ubiquitously in all known terrestrial ecosystems in symbiotic associations within the root system of most plants (Smith and Read, 2008), are studied for their potential benefits in sustainable agricultural production (Rooney et al., 2009).

Arbuscular mycorrhizal fungi show potential role in growth promotion by improving nutrition and disease resistance (Demir and Akkopru, 2007). Among the reported underlying mechanisms of AM interaction in plant resistance against pathogens are the specific competition against pathogen, root alterations and other defense related responses (Wu et al., 2013). Thus, the aim of this investigation was to determine the role of Glomus fasciculatum in the biological control of S. rolfsii and identify the defense related responses triggered by the AM-fungi in groundnut cultivar JL-24 sown on sterile soil.

MATERIALS AND METHODS

Plant and fungal material

Four groundnut seeds (Arachis hypogaea L.) of a local susceptible cultivar (var. Phule Pragati-JL 24) were used in pots containing sterile soil. The pots were watered at regular intervals with no addition of chemicals or fertilizers in a greenhouse at the Department of Botany, University of Pune, India. In the greenhouse, the relative humidity was 30 %-70 %, the pots were adequately spaced, the dead leaves or debris were removed regularly and average daily temperature was 28 °C with a photoperiod of 12 h.

The pathogen S. rolfsii was isolated from the fields of Pune, Maharashtra, and was identified through the Division of Mycology, ARI, Pune. The pure culture of S. rolfsii was maintained on potato dextrose agar (PDA) medium. The pathogen inoculum was prepared by using sterilized sorghum seeds, which were inoculated with pure culture of S. rolfsii and incubated for three weeks. This served as pathogen inoculum and after 15 d of plant growth a treatment set was inoculated with 5 g around the roots of groundnut plants. The control treatment received sterilized soil instead of pathogen inoculum.

Observations

The plants were harvested after 30, 60 and 90 d of planting (DAP) and variables measured were shoot length, root length, pod number, leaf number, fresh weight and dry weight. The severity of disease was measured by using a 0-5 scale: 1 = healthy plant; 2= lesions on stems only; 3 up to 25 % of the plant symptomatic (dying, wilted and dead); 4= 26-50 % of the plant symptomatic; 5= 50 % of the plant symptomatic (Shokes et al., 1996). The variables determined were: percent of root colonization of G. fasciculatum by using the Grid-line intersects methods (Giovannetti and Mosse, 1980), mycorrhizal dependency (Plenchette et al., 1983), total chlorophyll content (Arnon, 1949), total phenols (Malick and Singh, 1980), protein content (Lowry et al., 1951), polyphenol oxidase (PPO) activity (Mahadevan and Shridhar, 1982), and antioxidant enzyme activity of peroxidase (PER) (Putter, 1974).

Experimental design and statistical analysis

The experimental design was completely randomized block, with four treatments: 1) Control uninoculated (C); 2) inoculated with pathogen S. rolfsii (Sr); 3) inoculated with AM fungi G. fasciculatum (Gf); 4) inoculated with G. fasciculatum and S. rolfsii (Gf+Sr). Data were used to perform a one-way ANOVA and treatments means were compared using Tukey test (p≤0.05). The values are the mean of three replications ± S.E. (four plants per pot).

RESULTS AND DISCUSSION

Growth variables

The results (Table 1) show that after 60 and 90 d of growth, the morphological variables shoot, root length, fresh, dry weight and pod and leaf number were significantly increased by inoculating the groundnut plants with G. fasciculatum (Gf), as compared to non-inoculated control (C). In the presence of S. rolfsii, the G. fasciculatum inoculated groundnut plants (Gf+Sr) showed significant increase in growth variables as compared to non-mycorrhizal diseased (Sr) plants. The marked increase in growth response of groundnut plants inoculated with G. fasciculatum may be explained by its role in improvement of nutritional benefits (Smith and Read, 2008). Also, growth responses by G. fasciculatum inoculation in the presence of S. rolfsii (Gf+Sr) were increased, which would be due to the ability of AM fungi for inducing various defense resistance against pathogen infection (Cameroon et al. , 2013), irrespective of nutritional benefits of the AM fungi. That is why the increased growth responses by inoculation with G. fasciculatum signify their role in growth promotion in groundnut plants.

The symptoms of disease severity were reduced by 50.00 % in groundnut plants (Gf+Sr) treated with G. fasciculatum, as compared to non-mycorrhizal control ones (80.56 % in C+Sr) after 90 d of plantation. The severity of disease showed continuous reduction due to mycorrhizal inoculation in groundnut plants, as compared to non-inoculated diseased control (Figura 1). The mechanism of AM fungi in reducing severity of disease might be due to competition for resources or spaces with the pathogens (Maherali and Klironomos, 2007). The AM fungi establish themselves in the roots of host plants and the increase in root length of mycorrhiza treated groundnut plants may also be correlated with reduction of disease severity.

Percent root colonization

The G. fasciculatum inoculation resulted into formation of typical mycorrhizal structures such as arbuscules and vesicles, which was their establishment. Thus, the percent root colonization was highest by 90.00±3.27a % only in G. fasciculatum treated groundnut plants (Gf) as compared to 52.67±9.53b % in presence of pathogen (Gf+Sr) after 90 d of planting (Figure 2). The results showed that the pathogen S. rolfsii significantly reduced root colonization to 41.00±4.32b % (Gf+Sr) after 30 d as compared to 58.33±2.36a % in Gf treatment. This reduction may be due to the mechanism of competition among G. fasciculatum and S. rolfsii in groundnut plants.

Mycorrhizal dependency

The range of mycorrhizal dependency was higher by 54.40±5.69a after 30 d, 58.34±1.83a after 60 d and 69.29±0.94a after 90 d for G. fasciculatum inoculated groundnut plants in presence of pathogen (Gf+Sr), compared to healthy mycorrhizal plants (38.42±13.71ab after 30 d, 42.14±6.52b after 60 d and 56.17±2.75b). But, in the absence of pathogen the mycorrhizal dependency showed lower range of 38.42 - 56.17 % (Figure 3). The results showed the significance of G. fasciculatum during biotic stress caused by S. rolfsii. The result in terms of the dependence of groundnut plant on mycorrhizal associations for protection against pathogen may be correlated to their competitor establishment in plant roots (Harrier and Watson, 2004). Besides, the application AM fungi decreased disease severity (Figura 1).

Total chlorophyll

Chlorophyll absorbs and utilizes light energy during photosynthesis and the photosynthetic ability of plants are regulated (Taiz and Zeiger, 2002). In our experiment there was more total chlorophyll in mycorrhizal healthy groundnut plants (Gf) as compared to non-mycorrhizal diseased (C+Sr) or control after 30, 60 or 90 d of planting (Figure 4). The chlorophyll content was lowest in non-mycorrhizal diseased groundnut plants (C+Sr) but treatment with G. fasciculatum (Gf+Sr) increased the chlorophyll content in spite of pathogen's presence. The results may also be related with the increased number of leaves by mycorrhizal treatment, which may influence the rate of photosynthesis.

Biochemical variables

The protein content was highest in mycorrhizal diseased groundnut plants (Gf+Sr) and the healthy mycorrhizal groundnut plants (Gf) showed more protein than non-mycorrhizal control (C) (Table 2). The presence of pathogenesis-related (PR) proteins in plant tissues is associated as a mechanism of host plants for inhibiting pathogens (Wasternack et al., 2006). Hence, the increased protein content in our study may be due to pathogen invasion and the colonization by G. fasciculatum may have assisted in this defense in the groundnut plants.

This study shows (Table 2) different levels of total phenol content due to S. rolfsii and G. fasciculatum inoculations. The total phenols were highest in diseased mycorrhizal groundnut plants (Gf+Sr) followed by single inoculation of G. fasciculatum (Gf) and S. rolfsii (C+Sr). Here, the higher level of phenolics in mycorrhizal plants indicates the role of AM fungi in cell wall reinforcement as defense response against S. rolfsii infection. Moreover, the mycorrhizal associations induce cell wall bound phenolic acids for regulation of plant defense mechanism (López-Ráez et al., 2010).

The PPO showed higher activities in diseased non-mycorrhizal groundnut plants (C+Sr) than healthy mycorrhizal plants (Gf) (Table 2). The PPO activities were highest in mycorrhizal diseased plants (Gf+Sr). The reason for higher PPO activity is their involvement in oxidation of phenolic compounds into more toxic compounds, which creates adverse effects on the development of pathogens (Mohamed et al. , 2012). Moreover, the PPO activities were observed in mycorrhizal associations for the inhibition of pathogen (Jaiti et al., 2008).

The antioxidant enzyme (PER) in leaves of groundnut plants was higher in diseased non-mycorrhizal groundnut plants (C+Sr) when compared to healthy mycorrhizal (Gf) or control (C) (Table 2). The PER enzyme was highest in diseased mycorrhizal groundnut plants (Gf+Sr) as compared with non-mycorrhizal diseased or healthy mycorrhizal or control ones. The elevated PER activity indicates that oxidative burst may have occurred due to the attack of pathogen S. rolfsii in groundnut plants. As a result, reactive oxygen species (ROS) should be a defense mechanism, but excessive ROS accumulation may damage DNA, proteins or lipids (Mendoza, 2011). Therefore, the increase in PER enzyme was observed in groundnut plant, which protects from ROS damage.

CONCLUSIONS

The AM fungi (G. fasciculatum) significantly reduced disease caused by the soil-borne pathogen (S. rolfsii) in the groundnut cultivar (JL-24). Moreover, the increased growth responses, physiological, biochemical and anti-oxidative enzyme activities of chlorophyll, protein, total phenol, PPO and PER enzyme due to inoculation with AM fungi against pathogen S. rolfsii refers their beneficial role played in increasing the growth of plant as well as in bringing about possible induction of defense related activities. Thus, this experiment suggests the significance of mycorrhizal inoculation in bio-control of soil-borne pathogen in eco-friendly way.

ACKNOWLEDGMENTS

This research was funded by University Grants Commission, New Delhi, India, Rajiv Gandhi National Fellowship (No. F. 14-2(SC)/2009(SA-III).

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