Induction of Resistant to Radopholus similis and Defence Related Mechanism in Susceptible and Resistance Banana Hybrids Infected with Radopholus similis

Bananas and plantains (Musa spp.) are the second largest fruit crop produced and exported in the world and ranking third in terms of total production (Dochez et al., 2006). Plant parasitic nematodes are one of the major biotic stresses affecting banana production. Among them, the burrowing nematode, Radopholus simili is found in all major banana growing regions of the world (Hölscher et al., 2014). This migratory endoparasitic nematode causes root and corm tissue cavities that evolve to form necrotic lesions that affect the ability of the plant to uptake water and nutrients, resulting in the reduced development of banana bunches, reduced fruit yield, toppling and also paving way to pathogenic microorganisms (Aravind et al., 2010 and López-Lima et al., 2013).


Introduction
Bananas and plantains (Musa spp.) are the second largest fruit crop produced and exported in the world and ranking third in terms of total production (Dochez et al., 2006). Plant parasitic nematodes are one of the major biotic stresses affecting banana production. Among them, the burrowing nematode, Radopholus simili is found in all major banana growing regions of the world (Hölscher et al., 2014). This migratory endoparasitic nematode causes root and corm tissue cavities that evolve to form necrotic lesions that affect the ability of the plant to uptake water and nutrients, resulting in the reduced development of banana bunches, reduced fruit yield, toppling and also paving way to pathogenic microorganisms (Aravind et al., 2010 andLópez-Lima et al., 2013).
Crop losses by nematodes to banana are estimated to be very high, with an average annual yield loss of about 20 per cent worldwide . In addition, these parasites also interact with

ISSN: 2319-7706 Volume 6 Number 4 (2017) pp. 1668-1684
Journal homepage: http://www.ijcmas.com The burrowing nematode, Radopholus similis is considered to be the most destructive nematode associated with banana growth worldwide. Cultural and chemical management alone cannot guarantee full control. An alternative is to develop new banana hybrids with resistance to burrowing nematode. The twenty four new synthetic banana hybrids were screened under artificially inoculated pot condition for their reaction and resistance mechanism against Radopholus similis. Five hybrids namely, H 912, H 914, H 916, H 926, H 943 were rated resistance to Radopholus similis based on the nematode multiplication, lowest root lesion and corm lesion index. The nematodes inoculated resistant hybrid H 916 had higher total phenols (567.21 μg/g) and orthodihydroxy phenols (2.77 μg/g) activities than the susceptible cultivars. The enzymes changes in root content of peroxidase (2.78-3.32 abs/min/g), polyphenol oxidase (0.086-0.113 abs/min/g) and phenylalanine ammonia lyase (23.28-30.12 nmol/min/ml) of the hybrids in defense mechanism in response to nematode invasion indicated higher activities in resistance plants viz-a-viz susceptible ones.
other disease causing organisms of fungus Fusarium oxysporum f. sp. Cubense produce wilt disease complexes (Begum et al., 2012). Chemical control is widely used to manage this nematode, but this has become highly unsustainable due to high costs, deteriorating soil health, ground water contamination, hampering non target organisms, residue in fruits and general environmental issues (López-Lima et al., 2013). Therefore, host plant resistance has been recognized as one of the most economic, effective and environmentally-friendly measures for controlling the Radopholus similis nematodes.
Breeding of banana varieties with resistance to nematodes are considered a more sustainable management option, equally accessible for subsistence banana growers as well as commercial producers. The use of resistant cultivars is considered one of the most effective and environmental-friendly alternatives as nematode reproduction is reduced, no toxic residues in fruits, stabilizes the yield and blends with cultural control (Olowe, 2007;Moon et al., 2010). A resistant plant restricts or prevents the nematode's reproduction by activating defense mechanisms which may limit penetration of second-stage juveniles, inhibit of feeding site and prevents the reproduction of the adult female (Rodrigo et al., 2013). The identification and use of resistant or tolerant varieties can be a viable means of minimizing loss caused by nematodes (Gharabadiyan et al., 2012). The genetic component of host management involves the identification and utilization of selected sources of resistance in the breeding programs for development of nematode resistant cultivars (Hussain et al., 2014). Resistance can be considered as the ability of the plant to suppress development of pest and pathogens, whereas tolerance is the ability of the plant to grow well despite infection by pathogens .
The resistance and susceptibility attributes of several crops to different insect-pests and pathogens has been assessed with the presence of secondary plant metabolites mainly phenols (Vandana Sukhla et al., 2014;Pathipati and Yasur, 2010). The phenolic accumulation was increased by 56% in banana cv. Nendran after nematode infection whereas there was only 2% increase in cv. Karthobiumtham (Sundararaju and Pandi Suba, 2006). The biochemical contents like total phenols and lignin of the banana hybrids in defence mechanism in response to nematode invasion indicated higher activities in resistance genotypes viz-a-viz susceptible ones (Kavitha et al., 2008). Polyphenol oxidase, phenylalanine ammonia lyase enzymes activities and total phenols contents in roots were higher in nematode resistance banana hybrids than in the susceptible hybrids (Das et al., 2013(Das et al., , 2014. Phenol content had increased upto 50% at 30 days after sowing, which protected the plant from the pest by imparting high level of resistance (Taggar et al., 2014). Moreover, high levels of two major oxidizing enzyme of plants such as poly phenol oxidase and peroxidase impart induced resistance to insect herbivores and pathogens (Rachana et al., 2015;Bandi and Siva subramanian, 2012;He et al., 2011). Similarly, polyphenol oxidase and peroxidase activities were higher in resistant genotypes compared to susceptible genotypes of Capsicum annum infested by Bemisia tabaci (Latournerie-Moreno et al., 2015). Higher activity of peroxidise and polyphenol oxidase in faba bean was strongly associated with its resistant character against aphid Aphis craccivora (Soffan et al., 2014). Induction of polyphenol oxidase activity in potato leaves resulted due to aphid Myzus persicae infestation led to enhancement of resistance in potato against the pest (Xiao-Lin et al., 2013). Plants when attacked by R. similis nematodes show selective changes in their metabolism due to host pathogen interaction, inducing an immune response of host the parasite. Hence, the present investigation was undertaken to screen the banana hybrids for resistance against Radopholus similis nematode which can be further used as resistance source.

Materials and Methods
Twenty four new synthetic banana hybrids and their parents were derived from hybrid maintained block in TNAU Orchard, were studied at Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, during 2011-2013. Healthy sword banana suckers of uniform size and weight (750 g) were collected, pared, treated in hot water (50-55°C) for 10 minutes and planted, in plastic bag (30 x 45 cm) containing twenty kilograms of pot mixture (red soil: sand: FYM at 2:1:1 respectively) sterilized with 4% formaldehyde. The individual pots were labeled with name of the hybrid. The experiment was conducted in a glass house in potted plants, which were artificially inoculated with nematodes. Four weeks after planting, 10 plants of each hybrid were inoculated with nematodes while another set of 10 plants were kept as nematode-free control. The experiment was laid out in completely randomized block design. The hybrids were evaluated along with the reference cultivar viz., Yangambi km5 as the resistance cultivars and Grand Naine as the susceptible reference cultivar.

Culturing and extraction of nematodes and Inoculation of nematodes in pots
Healthy banana corms of cv. Robusta were selected and pared with a knife before planting to remove the outer layer of adhering roots and tissues to eliminate nematode infection. Sucker planted at the rate of one per pot, filled with autoclaved pot mixture consisting of sand, red soil and FYM mixed in equal proportions. Roots infested with R. similis were collected from infested field of banana, washed in water, cut into small bits and processed in a warring blender. The nematodes were extracted and the nematode suspension was then poured into the rhizosphere of the plants after the emergence of roots. Banana hybrids maintained in the pots were inoculated with infective juveniles of burrowing nematode, R. similis at 45 days after planting @ 1,000 nematodes /pot, respectively. The nematodes were extracted by the modified baermann funnel technique (Schindler, 1961). The nematode suspension was then poured in the holes made around the rhizosphere of the plants after the emergence of roots i.e. at 45 after planting . After inoculation the soil was lightly watered. Biochemical estimation was done in roots at 90 th day after inoculation.

Observations in pot culture
The nematode population in roots, root lesion index, corm grade and biochemical activity in roots were assessed 90 days after inoculation of nematode. To estimate the population of the nematodes, the roots of the inoculated plants were washed free of soil, dried by wrapping in absorbent tissue and cut into pieces of 1cm and weighed. Then, a sub sample of 15 g of roots per replicate was put into 100 ml distilled water in a kitchen blender and macerated 3 times for 10 sec and sieved through 250-106-40 µm sieves. The nematodes from the 40 µm sieve were collected in a beaker and made to a standard volume of 200 ml. The suspension was agitated with a pipette and 6 ml was taken in a counting dish to count nematodes under a stereomicroscope (Carlier et al., 2003).
The extent of nematode damage to roots and corms was assessed following the technical guidelines prescribed by INIBAP (Pinochet, 1988). Plants were removed from the pots and the soil washed from the roots and corm with tap water. Roots were collected from plants were divided into dead and functional roots. The percentage root necrosis was estimated for five randomly selected functional primary roots. Five primary root segments of 10 cm were cut lengthwise and the percentage of visible necrotic cortical tissue of five root halves was determined. Each root half could have a maximum percentage root necrosis of 20%, adding up to 100% for the five root halves together (Speijer and Gold, 1996;Speijer and De Waele, 1997). Corm damage assessment was done after thoroughly shaking off all soil and washing the corms with water. The number of root showing black -purple lesions around their bases on the selected corm was counted and scored for nematode related damage, which appears as blackish purple lesions around the root bases and was scored as: 0 = no lesions: 1= one small lesion, 2= several small lesions, 3= one large lesion and 4 = several large lesions (Pinochet, 1988) was followed to the hybrids as resistance, tolerant or susceptible as described in table 1.

Assessment of biochemical changes
The content of the biochemical phenols, lignin and orthohydroxy phenols and content of peroxidase, polyphenol oxidase and phenylalanine ammonia lyase in the root were determined for each replicate after 90 days, just before root samples were scored for nematode damage. The total phenol in the roots was estimated using Folin-Ciocalteau reagent and measuring absorption at 660 nm in a spectrophotometer, and is expressed as mg/g root (Spies, 1955) and Ortho-dihydric phenol by Arnow's method (Arnow, 1937). The lignin content of banana roots was gravimetrically estimated methods of Chesson (1978). For enzyme extraction, one gram of root sample per replicate was homogenized with 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) at 4 o C. The supernatant was used as crude enzyme extract for assaying peroxidase and polyphenol oxidase. Enzyme extracted in borate buffer was used for estimation of phenyl alanine ammonia lyase. The peroxidase activity was assessed according to Hammerschmidt et al., (1982) and polyphenol oxidase activity was assessed using the modified method of Mayer et al., (1965). Data were subjected to analysis of variance using the SPSS 20 statistical package and means compared by the LSD at P= 0.05.

Results and Discussion
Nematode population densities and root necrosis index were the most discriminate in this study on showing the difference in host reaction by banana hybrids to burrowing nematodes. Significant differences were observed among the hybrids for root population of R. similis at 90 th days after nematodes inoculation (Table 2). In all experiments, the mean average number of nematodes in roots of R. similis was low on the resistant reference cultivar Yangambi km5 (104 nematodes/5 g of roots) and high on the susceptible reference cultivar Grand Naine (417 nematodes/5 g of roots population ranges from 167 to 344. Some of the tolerant plant had higher nematode population but the growth of plant was not affected. This could be because, these plants allowed entry of the nematodes and their reproduction, but did not support further nematode growth. Similar results were also observed by Ramesh kumar et al., (2012) and Das et al., (2014).
Based on the intensity of lesions on roots and corm, they were assessed for their level of resistance ( The damages caused by R.similis on the banana root system of resistance cultivars were always lower than on susceptible cultivars, from 10.5-19% to 48-56% (Dochez et al., 2006). Root necrosis was a useful parameters in rating host resistance or susceptible to R. similis (  (1) while Grand Naine recorded maximum root lesion index (5) and corm grade (4). Good root development with healthy roots and corm favours resistance. Among the hybrids, 5 exhibited resistance, 10 exhibited tolerance, 5 were moderately susceptible and 4 were highly susceptible to nematode infestation. However, the use of dead roots percentage, nematode density and number of large lesions appears effective and an efficient approach to identify nematode resistant genotypes than the root necrosis index (Hartman et al., 2010).
The lower percentage of root lesion index and corm lesion index in the resistance hybrids might be due to lower nematode population, less multiplication rate in soil and roots. Similar finding were earlier reported by Das et al., (2010). The resistance banana cultivars had lower density of nematodes and least root damage. They may be considered as partially resistance to Radopholus similis (Gaidashova et al., 2008). The resistance cultivar Gros Michel had lower root infestation reflect a lower nematode carrying capacity, probably linked to its lower sensitivity related to secondary metabolism (Quenneherve et al., 2009).
The resistance hybrids exhibited its roots higher phenolic content and lignified cells also confirmed by histological studies. Carlier et al., (2002) reported that assessment of root and corm damage will give a better understanding of resistance or tolerance of the cultivars under both field and glass house conditions. Banana varieties Gros Michel and Culcatta 4 were much lower root necrosis, percentage of dead roots and population densities which were considered partially resistance to R. similis nematode .
Several physiological processes in the host are stimulated due to the activation of certain enzymes. Resistance to nematode is often governed by the intrinsic capability of the cells to deter the movement and feeding or interrupt the nematode reproduction by way of synthesis of certain chemical substances. Many of these proteins are enzymes such as phenylalanine ammonia lyase, polyphenol oxidase, peroxidase and b-1-3 glucanase. These are involved in the synthesis of low molecular weight substances such as phytoalexins, phenols and lignin, which are inhibitory to the invading nematodes (Seenivasan et al., 2012).
Phenolic compounds are known to play a major role in the defense mechanism of plants against various external infectious agents. The total phenol content of the banana hybrids was estimated in roots and the results showed that there was a significant difference among the hybrids, treatments and their interaction (  Fogain and Gowen (1996;Damodharan (2007), Kavitha et al., (2008), Karunakaran (2010) Das et al., (2014) and Ranchana et al., (2016). The plant infected by nematodes and the accumulation of phenolic compounds in the plants (Selim et al., 2014). Many of these compounds, especially oxidized forms, are toxic to repelling the juveniles or by adversely affecting the development of juveniles nematode and act as mechanical barriers to nematodes enter into the plant.
Increase in phenol content and enzyme activities were negatively related with the degree of infestation. The accumulation of phenol may be due to the excess production of hydrogen peroxide by increased respiration (Farkas and Kraly, 1962) or due to the activation of hexose monophosphate shunt pathway, acetate pathway and release of bound phenols by hydrolytic enzymes (Goodman et al., 1967;Seenivasan, 2011). Fogain 1996 andValette et al., (1997) found that higher amount of phenolics in the resistance banana cultivar of Yangambi km5. Most of the nematode resistance plant is found in hypersensitive type of response that involves change in enzyme activity, phenol metabolism and deposition of the newly synthesized material in cell walls and regulation of free redical O 2 (Ganguly and Dasgupta, 1980;Zacheo et al., 1995). Similar results were also observed by Das et al., (2011) for nematode resistance in banana.
Irrespective of the hybrids screened, the nematodes inoculated plants registered higher Orthodihydroxy phenol content in roots compare to uninocualed hybrids (Table 3). The highest Orthodihydroxy phenol content was observed in the hybrids, H 916 (2.77 g/g) which showed an increase of 48.92 per cent over the control. The OD phenol was found to be lower (0.98 g/g) in H 922 which showed an increase of 13.95 per cent over control. Studies on the changes in the Orthodihydroxy phenol clearly indicated that there was an enhancement of activities of this biochemical in the resistance hybrids.
Phenol and OD-phenol content had increased upto 50% at 30 days after sowing, which protected the plant from the pest by imparting high level of resistance (Taggar et al., 2014). The increased levels of orthodihydroxy phenols might have resulted as a means of defensive reaction to nematode infestation since orthodihydroxy phenols are known to be reactive and upon oxidation yield quinones which are still more toxic to invading organisms (Indu Rani et al., 2008). The per cent mean accumulation of orthodihydroxy phenol content in genotypes between before pathogen initiation stage to peak stage of infection was more in resistant genotypes (55.80%), than moderately resistant (46.27%) and susceptible (37.36%) genotypes (Sowmya, 2011).
The infestation due to nematodes increased the lignin in all banana hybrids compared to uninoculated plants and the differences were significant. Among the hybrids screened, uninoculated hybrids (control) registered the highest lignin was registered in H 926 (1.12 per cent) while lowest was recorded in H 940 (0.51 per cent) (Table 3). In nematode inoculated hybrids, H 912 registered the highest lignin content of 1.93 per cent. The percentage increase of lignin activity over control was more in the hybrid H 912 (78.70%).
Lignin is one of the most abundant biopolymers, which provides resistance to plants against R. similis and makes the cell wall more resistant to nematodes attack. The deposition of lignin around the vascular bundle has been implicated as a defense response in banana resistant cultivar to nematodes. Similar results were also observed by Kavitha et al., (2008) and Karunakaran (2010)

in banana.
A close relationship between lignification and disease resistance has been showed that resistant plants accumulated lignins more rapidly and/or exhibited enhanced lignin deposition as compared with susceptible plants (Yates et al., 1997). Lignin and phenol are synthesized via phenyl propanoid pathways which impart resistance against nematode attack. The role of phytoalexins and other toxic compounds like phenols and lignin in resistance mechanism have been reported by earlier workers (Reuveni et al., 1992 andSariah et al., 1999). An increase in the number of lignified cells in tolerant cultivars compared to susceptible cultivars of banana was noted by Fogain, (1996) and Kavino et al., (2007).
Enzyme activity is one of the important tools to confirm the resistance to root pathogenic nematodes. When a nematode infects the host tissue, a small number of specific genes are induced to produce mRNA's that permit synthesis of similar number of specific proteins (Vidhyasekaran, 1993). Many of these proteins are enzymes such as phenylalanine ammonia lyase, polyphenol oxidase, peroxidase and β-1-3 glucanase. These are involved in the synthesis of low molecular weight substances such as phytoalexins, phenols and lignin, which are inhibitory to the invading nematodes (Seenivasan et al., 2012) The infestation due to nematodes increased the peroxidase activity in all banana hybrids compared to uninoculated plants and the differences were significant (Table 4). The uninoculated hybrids, H 914 (2.47abs/min/g) registered the highest peroxidase content while the lowest was observed in H 941 (0.97abs/min/g). Among the inoculated hybrids, peroxidase activity of 3.32 abs/min/g fresh weight was highest in H 914 which showed 34.41 per cent increase over control conditions. The lowest activity of 1.16 abs/min/g fresh weight was recorded in H 922 with an increase of 10.48 per cent over control. Estimation of peroxidase activity in the current study elicits that all the resistant hybrids possessed higher peroxidase activity than the susceptible ones. A critical analysis of their activity in this study revealed that resistance hybrids viz., H 912, H 914, H 916, H 923 and H 943 recoded highest peroxidase activity than the susceptible hybrids. The percentage increase was more in resistant hybrids as compared to susceptible banana hybrids. Similar finding was also reported by Das et al., (2011) and Tapamay Dhar et al., (2016. Peroxidase activity in nematodes infected roots of tomato were considered, there total peroxidase activity was twice in resistance plants as compared to susceptible (Zacheo et al., 1993). Peroxidase makes cellular environment toxic and extremely unfavorable for pathogen by producing reactive species of oxygen and nitrogen (Passardi et al., 2005;Gill and Tuteja, 2010;Liu et al., 2010;Schaffer and Bronnikova, 2012). Peroxidase enzyme provide mechanism for resistance to pathogens, is highly essential Peroxidase enzymes also play a vital role in alleviating free radical toxicity in plant tissues (Fogain and Gowen, 1996;Valette et al., 1997;Elsen et al., 2002). Increased activity of peroxidase in tomato and phenylalanine ammonia lyase in brinjal was positively correlated with nematode resistance (Rajasekar et al, 1997;Sirohi and Dasgupta, 1993). Polyphenol oxidase and peroxidase, the enzymes involved in the oxidation of phenols to more toxic quinones, are known to increase in resistant plants (Yamamoto and Tani, 1978). Significant difference was noticed between the hybrids, treatments and the interaction with regard to polyphenol activity (Table 4). The hybrid H 923 expressed the maximum activity of 0.096 abs/min/g under control and 0.126 abs/min/g when inoculated while the minimum was in H 922 (0.032 abs/min/g) under control and (0.037abs/min/g) when inoculated. Increase in polyphenol activity was the highest in H 926 (41.56%) and the lowest in H 905 (9.30%) followed by H 901 (9.62%). Following inoculation of banana hybrids with the nematodes, the polyphenol activity increased in both the resistant and susceptible hybrids. However, the final enzyme concentration was the maximum in resistance hybrids like H 912, H 914, H 916, H 923 and H 943. Polyphenol activity was found to increase in all the hybrids following nematode inoculation. However, the final enzyme concentration was higher in the resistance hybrids. Increased peroxidase activity due to nematode infestation was reported by Fogain and Gowen (1996), Krishnamoorthy (2002) Peroxidase involved in defense mechanism considered the condensation of phenolic monomers derived from the phenylpropanoid pathway into insoluble polymers (Robb et al., 1991). Devarajan and Seenivasan (2002) observed that inoculation of nematodes increase the polyphenol oxidase activity in banana. It might have resulted in oxidation of polyphenols and accumulation of monophenols which are responsible for resistance reaction. Polyphenol oxidase (PPO) oxidizes the phenols to highly toxic quinones and hence is considered to play an important role in disease resistance, particularly those affecting the root tissues (Das et al., 2010). Thus, the overall estimation analysis of these enzymes in resistant and susceptible hybrids indicates the role of these enzymes in conferring resistance to nematodes.    The result of the study showed significantly lower activity of PAL in nematode inoculated plants of susceptible cultivars while in the resistance hybrids, the PAL activity increased in both healthy and nematode inoculated plants of hybrid (Table 4). Among the hybrids, H 904 registered the highest phenylalanine ammonialyase content of 24.00 nmol/min/ml in control and 29.67 nmol/min/ml under inoculated condition while H 905 registered lowest content of 9.51 nmol/min/ml and 10.33 nmol/min/ml under control and inoculated condition respectively. The maximum per cent increase in PAL was recorded by H 926 (38.67 nmol/min/ml), while the lowest by H 905 (8.62 nmol/min/ml). Phenylalanine ammonia lyase is the most important enzyme in the synthesis of phenolics, phytolexin and lignin. Hence it is considered as the most important enzyme in nematode resistance.
The phenylalanine ammonia lyace activity in resistance diploid accessions of banana roots had higher activity after nematode inoculation compared to susceptible banana   Das et al., (2014). PAL is the first enzyme in phenylpropanoid metabolism involved in the production of phenolics and phytoalexins that prevent establishment of the pathogen (Mariutto et al., 2011). This enzyme induces the formation of necrosis, which resulted in the localization of the juveniles and prevented their further movement. The maximum activity of glucanase, chitinase and PAL was found to be associated with resistant status of mungbean cultivars (Deepak and Choudhary, 2012). The overall evaluation of 24 banana hybrids led to identification of the hybrids H 912, H 914, H 916 H 926 and H 943 were resistant to R. similis. These studies indicate that higher content of phenols, lignin and PO, PPO and PAL might have produced toxic compound against nematodes. These resistance hybrids can be utilized in future breeding programme for developing nematode resistance hybrid. Indeed, the highest yielding hybrids evaluated in multilocation trials and could be released for cultivation.