Interaction between Meloidogyne incognita and Rhizoctonia solani on green beans.

The interaction between Meloidogyne incognita (race 2) and Rhizoctonia solani (AG 4) in a root rot disease complex of green beans (Phaseolus vulgaris) was examined in a greenhouse pot experiment. Three week-old seedlings (cv. Contender) were inoculated with the nematode and/or the fungus in different combinations and sequences. Two months after last nematode inoculation, the test was terminated and data were recorded. The synchronized inoculation by both pathogens (N + F) increased the index of Rhizoctonia root rot and the number of root galls; and suppressed plant growth, compared to controls. However, the severity of root rot and suppression of plant growth were greater and more evident when inoculation by the nematode preceded the fungus (N → F) by two weeks. Nematode reproduction (eggs/g root) was adversely affected by the presence of the fungus except by the synchronized inoculation. When inoculation by nematode preceded the fungus, plant growth was severely suppressed and roots were highly damaged and rotted leading to a decrease of root galls and eggs.

Introduction

Green beans (Phaseolus vulgaris L.) is a very important summer vegetable crop in Saudi Arabia, and grown in the open fields and greenhouses mainly for its green pods. The crop is frequently attacked by Meloidogyne javanica (Treub), Chitwood and Meloidogyne incognita (Kofoid & White) Chitwood (Al-Hazmi, 1985; Al-Hazmi et al., 1995). Green beans are also very susceptible to Rhizoctonia solani Kuhn (Hall, 1991). In a field survey of fungal pathogens associated with green beans in the central region of Saudi Arabia, 17 species of pathogenic fungi were recorded (Al-Osaimi, 2005). Among these fungi, R. solani was found to be the second most common species and the most severe on green beans. The fungus R. solani was found frequently associated with the root-knot nematodes. Interaction between these two pathogens in our field soils might play a very damaging role in our green bean fields.

Since the first recorded case of nematode–fungus interaction in 1892 (Atkinson, 1892), interest in such interactions and their damages to many economic crops has been attracting many scientists. Interactions between the root-knot nematodes (Meloidogyne species) and the root rot fungus R. solani have been studied and documented in several host crops including green beans. Several reviews on the subject have been published (Powell, 1971; Back et al., 2002; Shahzad and Ghaffar, 1992; Mai and Abawi, 1987; Evans and Haydock, 1993).

Several reports indicated that Rhizoctonia-root rot was more severe in the presence of root-knot nematodes, including the root rot disease complex caused by R. solani and M. incognita on green beans (France and Abawi, 1994; Mokbel et al., 2007; Abuzar, 2013; Ali and Venugopal, 1992; Batten and Powell, 1971; Chahal and Chhabra, 1984; Shahzad and Ghaffar, 1995; Sharma and Gill, 1979; Anwar and Khan, 2002; Reddy et al., 1979; Bhagwati et al., 2007). Most of these reports indicate a synergistic interaction between these two important pathogens. Rhizoctonia-root rot generally affects seedlings, but fungus can also infect mature plants and induce root rot leading to plant wilt and finally death of infected plants.

The objective of this present study was to evaluate the interaction of M. incognita (race-2) and a local isolate of R. solani in a root rot disease complex of green beans (cv. Contender) under the greenhouse conditions.

Materials and methods

This greenhouse experiment consisted of individual, concomitant and sequential inoculation of M. incognita (race-2) and R. solani (AG4). The test included six treatments, namely: (1) M. incognita alone (N); (2) R. solani alone (F); (3) both pathogens simultaneously (N + F); (4) nematode first and then fungus two weeks later (N → F); (5) fungus first and then nematode two weeks later (F → N); and (6) control (non-inoculated seedlings) (Table 1).

Table 1

Effects of Meloidogyne incognita (N) and Rhizoctonia solani (F) singly and combined on the severity of root rot of green beans.

Treatment⁎	% root area infected	Root rot dis. index (0–4)⁎⁎	% Severity of root rot⁎⁎⁎
F	51 bc	2.2 c	52 bc
N + F	58 b	2.8 b	58 b
N → F	84 a	3.6 a	82 a
F → N	42 c	2.0 c	42 c

Values are means of five replicates. Means, in each column, followed by the same letter (s) are not significantly, different at P ⩽ 0.05.

Sequences: N + F = simultaneous inoculation, N → F = nematode applied 2 weeks before fungus, F → N = fungus applied 2 weeks before nematode.

On a scale of 0–4, where 0 = healthy; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%, and 4 = 76–100% root area is infected (Aoyagi et al., 1998).

(Aoyagi et al., 1998).

The nematode inoculum consisted of eggs which were extracted in 0.05% sodium hypochlorite (Hussy and Barker, 1973) from a pure greenhouse culture of M. incognita (race-2) on tomato plants. The egg suspension was immediately washed several times with sterilized distilled water, and then, adjusted to contain 1200 eggs/ml of the suspension. The nematode inoculum used was 12,000 eggs/seedling.

A pure culture, on Potato Dextrose Agar (PDA), of R. solani originally isolated from green bean plants from local fields, was obtained from the Mycology Unit (Dr. Saleh El-Hussaini), laboratory of fungal plant diseases, Department of Plant Protection, King Saud University, Riyadh, Saudi Arabia. The fungus was, then, maintained on PDA in petri plates (at 25–27 °C) for a week. For inoculum preparation, 250 ml conical flasks, each containing about 10 g of barley grains soaked overnight in sterilized distilled water, were used. The media in flasks were autoclaved for 30 min in two consecutive days. After the flasks were cooled, each one was inoculated with a small block (5 mm diam.) taken from the periphery of the 7-day-old cultures on PDA. The flasks were, then, incubated at 27 ± 2 °C for two weeks. During incubation, the flasks were shaken twice a day to ensure the proper growth of fungal mycelium on the barley seeds. The fungal-colonized barley seeds were used as inoculum at the rate of 15 g/seedling.

The green bean (Phaseolus vulgaris L.) cultivar used in this study was “Contender” which is known to be susceptible to both M. incognita (race-2) and the fungus R. solani. Uniform 3-week-old seedlings were transplanted singly into sterilized plastic pots (14 cm diam.) containing a steam-sterilized mixture (1500 g soil) of equal parts of sand, soil, and peat moss. The seedlings were fertilized with Hogland’s solution and left in the greenhouse for two weeks before treatments.

At inoculations with nematode and/or fungus, each seedling was inoculated with 12,000 eggs and/or 15 g of the fungus inoculum on barley grains depending on the designated treatment (Table 2). Non-inoculated seedlings served as control. The nematode egg inoculum, suspended in 10 ml of water, was equally distributed through three small holes made in the soil around the seedling stem and deep enough to contact the roots. Inoculation with the fungus was made by distributing and mixing the fungal inoculum thoroughly with the soil surface of the designated pots. Each treatment was replicated five times, and treatments were arranged on a bench in the greenhouse (25–27 °C) in a completely randomized design. All seedlings were irrigated and fertilized with Hogland’s solution as needed.

Table 2

Effects of Meloidogyne incognita (N) and Rhizoctonia solani (F) singly and combined on host response of green beans.

Treatment⁎	Total plant weight (g)	% Change	No. of root galls/g root	% Change
Control	9.7 a	–	0.00	–
N	5.9 b	−38.6	91.0 c	–
F	6.6 b	−32.0	–	–
N + F	5.3 b	−45.8	192.0 a	+111.0
N → F⁎⁎	2.18 c	−78.0	71.2 c	−22.0
F → N	5.4 b	−44.4	143.0 b	+57.2

Values are means of five replicates. Means followed by the same latter are not significantly different at P ⩽ 0.05.

Sequences: N + F = simultaneous inoculation, N → F = nematode applied 2 weeks before fungus, F → N = fungus applied 2 weeks before nematode.

Plants in this treatment were severely damaged.

Sixty days after inoculation, plants were uprooted, washed under tap water and growth parameters were recorded. Nematode infection was determined by the number of root galls and host growth, whereas the nematode reproduction was determined by the number of eggs on roots. Gall index (Taylor and Sasser, 1978) and reproduction factor (RF) of the nematode (Oostenbrink, 1966) were calculated. Root rot of each root system was determined according to four categories of root system necrosis: 0 = none; 1 = less than 25%; 2 = 26–50%; 3 = 51–75%; 4 = 76 = 100% (Aoyagi et al., 1998). Disease severity of root rot was also calculated according to the formula by Aoyagi et al. (1998).

Data were statistically analyzed (SAS, 2013), and treatment means were separated by protected Fisher’s least significant difference (LSD).

Results

Our results showed that root rot indices and disease severity (%) increased (P ⩽ 0.05) in plants inoculated with both pathogens (N + F) compared to the plants inoculated with the fungus alone (F) (Table 1). However, the greatest root rot disease was observed when the nematode preceded the fungal inoculation by two weeks

(N → F). This last increase was significantly higher than that of the simultaneous inoculation (N + F). The reciprocal treatment (F → N) did not increase the root rot disease compared to treatment of fungal alone (F) (Table 1).

The total fresh weights of plants inoculated with either or both pathogens were reduced (P ⩽ 0.05) compared to the non-inoculated plants (Table 2). This suppression of plant growth was more (P ⩽ 0.05) severe (−78.0%) when the nematode preceded the fungal inoculation by two weeks (N → F). In fact, plants in this treatment (N → F) were severely damaged and their very small roots were severely rotted and, subsequently, had lesser galls and egg production (Table 2). Number of root galls in the other two treatments (N + F, and F → N) were higher than the nematode alone (N) treatment (Table 2).

Reproduction of M. incognita was adversely affected (P ⩽ 0.05) when the fungus preceded the nematode inoculation (F → N) (Table 3). In the reciprocal treatment (N → F), the roots were severely damaged and had fewer eggs. The concomitant inoculation with both pathogens (N + F) showed similar nematode reproduction to that caused by the nematode alone (N) (Table 3).

Table 3

Effects of Rhizoctonia solani (F) on reproduction of Meloidogyne incognita (N) on green beans.

Treatment⁎	Eggs/g of root	Reproduction factor (RF)⁎⁎
N	241.07 a	129.6 a
N + F	236.07 a	131.0 a
N → F	18.52 c	11.0 c
F → N	57.68 b	30.4 b

Values are means of five replicates. Means, in each column, followed by the same letter (s) are not significantly, different at P ⩽ 0.05.

Sequences: N + F = simultaneous inoculation, N → F = nematode applied 2 weeks before fungus, F → N = fungus applied 2 weeks before nematode.

RF = Pf/Pi.

Discussion

Our results showed that the index of root rot caused by R. solani has increased in the presence of the root-knot nematode M. incognita. However, the severity of the root rot disease was more (P ⩽ 0.05) pronounced when the inoculation by the nematode preceded the fungus by two weeks (N → F). This increase of root rot indicates that the infection by both pathogens, whether simultaneously or the nematode first, would result in a synergistic interaction leading to a greater plant damage (1 + 1 > 2). This was supported by the reciprocal treatments when the fungus preceded the nematode (F → N) where the severity of the root rot was not significant compared to the infection by the fungus alone. The present results support previous similar reports of synergistic effects of R. solani and M. incognita, on different crops, such as mung beans (Shahzad and Ghaffar, 1995), cardamom (Ali and Venugopal, 1992), sunflower plants (Mokbel et al., 2007), cotton (Carter, 1981), okra (Bhagawati et al., 2007; Safiuddin and Shahab, 2012; Safiuddin et al., 2014), tomato (Chahal and Chhabra, 1984), chili (Abuzar, 2013), potato (Sharma and Gill, 1979), eggplant (El-Nagdi and Abd-El-Khair, 2008), grapes (Walker, 1994) and on other vegetables and field crops (Shahzad and Ghaffar, 1995). The increase of root rot severity in the presence of M. incognita may be due to the fact that the infection by this endo-parasitic nematode (M. incognita), whether simultaneously (N + F) or prior to the fungal infection (N → F), causes physiological and anatomical great changes in the root tissues leading to predisposing the plants to increased fungal infection (Carter, 1981; Powell, 1968; Porter and Powell, 1967; Batten and Powell, 1971). Giant cells induced by Meloidogyne spp. often make suitable sites for the pathogenic and non-pathogenic soil-borne fungi (Mai and Abawi, 1987; Mayol and Bergeson, 1970). All these changes caused by the nematode may explain the great increase of root rot and damage in the plants infected first by the nematode (N → F) compared to the plants in the other two treatments of fungus alone (F) or fungus first (F → N). Similar findings were reported by other researchers (Reddy et al., 1979; Shahzad and Ghaffar, 1992, 1995; Ali and Venugopal, 1992).

Root galls increased in both the synchronized treatment (N → F) and when the fungal preceded the nematode inoculation (F → N). This might be attributed to the increase of the penetration rate by J2 into the roots (Tu and Cheng, 1971). However, the plants inoculated with the nematode first (N → F) were much greatly damaged and their roots were severely rotted. Therefore, these roots became unsuitable for juvenile penetration and nematode development, and have much less galls and eggs.

Growth of plants inoculated with the nematode and/or the fungus was suppressed (32–78%), supporting similar previous findings (Reddy et al., 1979). However, the greatest suppression of growth (78%) was observed when the nematode preceded the fungal inoculation. Undoubtedly, the pathogenic physiological alterations caused by the nematode and/or the fungus are the main factor for this growth suppression. It has been also reported that green beans infected with M. incognita contain much less quantities of chlorophyll, carbohydrates and nitrogenous compounds; and show low capacity to absorb water and nutrients from soil, resulting in a growth and yield decrease (Melakeberhan et al., 1985; Wilcox and Loria, 1986).

The reproduction of M. incognita (eggs and RF) was suppressed by the presence of the fungus R. solani, especially in the two sequential treatments (F → N, and N → F). This finding agrees with previous similar reports on the adverse effects of several soil-borne fungi on the reproduction of Meloidogyne spp. on several crops (Mokbel et al., 2007; Al-Hazmi, 1985; France and Abawi, 1994; Griffin and Thyr, 1988; Sharma and Gill, 1979; Powell, 1971; Mousa and Hague, 1988). The great damage on roots caused by R. solani may have affected the nematode feeding process within the root tissues and, subsequently, adversely affected the nematode reproduction. Furthermore, the toxic metabolites produced by many pathogenic fungi may have deteriorated the giant cells which are necessarily for the nematode feeding and, then, reproduction, as well as the juvenile movement (Fattah and Wbester, 1989; Mokbel et al., 2007). These effective damages on nematode biology may explain the decrease of a number of eggs produced by the nematode.

Development of a successful strategy to manage this nematode/fungus disease complex should depend primarily on an applicable integrated disease management including suitable different methods to suppress the population of both pathogens in our field soils.