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African Crop Science Journal, Vol. 9. No. 3, pp. 527-535

STUDIES ON THE INTERACTION BETWEEN RALSTONIA SOLANACEARUM (SMITH) AND MELOIDOGYNE SPP. IN POTATO

E.M. ATEKA, A.W. MWANG'OMBE and J.W. KIMENJU
Department of Crop Protection, University of Nairobi, P.O Box 30197, Nairobi, Kenya

Received 9 March, 1999
Accepted; 30 March, 2001

Code Number: cs01072

ABSTRACT

A survey was undertaken to determine population density of Meloidogyne juveniles (J₂) in 90 fields randomly selected from three potato-producing districts in Kenya namely Nyeri, Meru and Nyandarua. Mean nematode densities were highest (45) in samples collected from Nyeri and Nyandarua and lowest (31) in soil samples collected from Meru. The reaction of 15 potato cultivars to Meloidogyne incognita was determined under greenhouse conditions. Plants were inoculated with 6000 eggs and second-stage juveniles each. Highly significant (P=0.01) differences were observed between the cultivars. Galling index was highest (5.5) in cv. KP93739.26 and lowest (1.9) in cv. Nyayo. All cultivars supported nematode reproduction with the highest (5.0) egg mass index being recorded in cultivars KP93739.26, Kerr's Pink, Desiree CIP-800048, KP92633.26 and B53. No cultivar exhibited immunity. The relationship between infection by root-knot nematodes and severity of bacterial wilt in three potato cultivars with varying levels of resistance to bacterial wilt namely Asante CIP 381381.20 (susceptible), B53 (moderately susceptible), and Kenya Dhamana (resistant), was investigated in a greenhouse experiment. Bacterial wilt was more severe in plants infected with both pathogens as compared to plants infected with R. solanacearum alone.

Key Words: Egg mass index, galling index, incidence, Meloidogyne, severity

Résumé

Une enqueté a été enterprise pour déterminer la densité de la population de jeunes Meloidogines (JM) dans 90 champs aléatoirement séléctionnés dans trois districts producteurs de la pomme de terre au Kenya à savoir Nyeri, Meru et Nyandarua. Les moyennes des densités des nématodes étaient plus élevées (45) dans les échantillons collectés dans Nyeri et Nyandarua et plus faibles (31) dans les échantillons de sols collectés dans Meru. La réaction de 15 cultivars de pomme de terre au Meloidogine incognita a été déterminée dans les conditions de serre. Les plantes étaient inocullées avec 6000 oeufs et le deuxième stade juvénile chacun. Des differences hautement significatives (P=0.01) ont été observées entre les cultivars. L'indexe de galles était plus élevé (5.5) chez le cultivar KP93739.26 et plus faible chez le cultivar Nyayo. Tous les cultivars ont supporté la production de nématodes avec l'indexe de masse des oeufs plus élevé chez les cultivars KP93739.26, Kerr's Pink, Desiree CIP-800048, KP92633.26 et B53. Aucun cultivar n'a montré d' immunité. La relation entre l'infection par les nématodes des noeuds des racines et la séverité du mildiou dans trois cultivars de pomme de terre avec different niveaux de résistance au mildiou à savoir CIP 381381.20 ( sensible), B53 ( modérement sensible), et Kenya Dhamana (résistant), a été étudiée dans un essai en serre. Le mildiou était plus sévère dans les plantes infectées par les deux pathogènes par rapport aux plantes seulement infectées par R. solanacearum.

Mots Clés: Indexe de masse des oeufs, indexe de galles, incidence, Meloidogyne, sévérité

INTRODUCTION

Potato (Solanum tuberosum L.) is the fourth most important food crop in the world after wheat, rice and maize with its production roughly representing half the world's annual output of all root crops and tubers (FAO, 1995). However, its cultivation is limited largely by diseases and pests (Hayward and Hartman, 1994).
Bacterial wilt was first reported in Kenya in the1940s and, since then, it has spread to most potato growing areas (Michieka, 1993). Over 50% yield losses have been reported in the country with occasional losses of up to 7 % on seed potato (Ajanga, 1993). The disease is a major constraint to potato production particularly because its management is difficult (Sequeira, 1992).
Root-knot nematodes (Meloidogyne spp.) are another major limiting factor to potato production worldwide (Bird, 1981). Their feeding reduces growth vigour and causes blemishes on tubers thus reducing their marketability (Sijmons et al., 1994). Losses resulting from root-knot nematode damage alone can reach 25% depending on the cultivar and prevailing environmental conditions (Mai et al., 1981).

Meloidogyne spp. occurs commonly in warm regions and, are therefore not a worldwide problem on potato because potatoes are mainly grown in cool regions (Evans and Trudgill, 1992). However, with increasing land pressure, production is expanding to the warmer lowlands where root-knot nematodes are undoubtedly troublesome.

The use of resistant cultivars has several advantages over other methods of reducing nematode populations; it requires little or no technology, it is cost effective and does not leave toxic residues (Trudgill, 1991). Nematicides, besides leaving toxic residues in the environment, provide partial control (Brown et al., 1989).

The influence of root-knot nematodes on the development and severity of bacterial wilt has been elucidated (Akiew et al., 1991). Plants infected with both Meloidogyne spp. and Ralstonia solanacearum have been found to express wilt symptoms earlier with greater severity than those infected with R. solanacearum alone (Johnson and Powell, 1969; Akiew et al., 1991). Sitaramaiah and Sinha (1985) have suggested that the nematode induced stress is more important as a wilt-triggering factor than as a wounding agent.

Whereas the reaction of most commercial potato cultivars to major diseases such as late blight (causal agent Phytophthora infestans), and bacterial wilt, Ralstonia (Pseudomonas) solanacearum, has been established, their reaction to root-knot nematodes is largely unknown. Additionally, potato reported as resistant in a given location may not be resistant to the same root-knot nematode species when grown in another location. In Kenya, the distribution and density of root-knot nematodes in potato growing areas is also unknown. Even though infection by Meloidogyne spp. is known to predispose potato plants to R. solanacearum, information on occurrence of the two pathogens and their interaction in Kenya is meagre. In addition, information is lacking on the impact of Meloidogyne infection on wilt expression in potato cultivars with varying levels of resistance to R. solanacearum.

The objectives of this study therefore were to (a) determine the occurrence of root-knot nematodes in Kenya, (b) assesse the interaction between bacterial wilt pathogen R. solanacearum and root-knot nematode Melodogyne spp. (c) determine the reaction of potato cultivars to M. incognita.

MATERIALS AND METHODS

Population densities of Meloidogyne spp. in potato fields. Thirty potato fields in Nyeri, Nyandarua and Meru district were randomly selected at intervals of 3-5 kilometers along the roads. The sampling procedure for nematode assessments was adopted from Dropkin (1980). Each potato field was divided into four blocks. From each block, five soil samples were taken from the rhizosphere of potato plants along a zigzag path. The top 5-cm soil layer was scrapped off before a sample of soil was obtained from a depth of 5-20 cm. Twenty soil samples were taken from each farm and mixed thoroughly before 3 kg of the composite sample was taken. Nematode extraction was done by the sieving and filtering technique (Hooper, 1990). Two-200 cm³ soil sub-samples were taken from the composite sample and placed in a bucket to which about 5 litres of water was added. The resulting suspension was stirred and passed through a 710 μm-aperture sieve nested on top of a 45 μm-aperture sieve. The residues on the 710 μm-aperture sieve were discarded while those on the 45μm-aperture sieves were backwashed into a beaker and then poured onto double milk filters supported on a screen standing in a shallow dish. Water was added into the dish until it was just touching the milk filters. Nematodes were allowed three days to move from the suspension, through the milk filters, into the water beneath. The resulting nematode suspension was concentrated by draining off excess water through four 45 μm-aperture sieves. The nematode suspension was adjusted to a volume of 10 ml by adding or siphoning excess water after allowing the nematodes to settle at the bottom of the vials. One-ml aliquot of a well-mixed suspension was drawn and pippeted into a counting slide and counting done under a light microscope. Counting was repeated in three aliquots to improve accuracy.

Resistance of potato to Meloidogyyne incognita.
Fifteen potato cultivars were evaluated for resistance to M. incognita in the greenhouse. The cultivars were Nyayo, Desiree CIP-800048, Roslin Tana, Kerrs Pink LB-5, Golof (Dutch Robjin), B53, Tigoni CIP-381381.13, Rutuku CIP-720097, Asante CIP-381381.20, Kenya Dhamana CIP- 800228, Mauritius Clone, KP93739.26, KP92633.26, Furaha and Cruza148 CIP-720118. Tomato (Lycopersicon esculentum L.) cv. Cal J was included as a susceptible control since there was no potato cultivar with known reaction to M. incognita. Potato tubers were sown in 20-cm diameter plastic pots containing heat-sterilized potting mixture of soil, sand and ballast at the ratio of 3:2:1. Inoculum consisted of 6000 eggs and second-stage juveniles of M. incognita suspended in 10 ml sterile distilled water. The inoculum was pipetted into 4-cm deep indentations made in the soil medium at the root zone of each plant, one-week after planting. Treatments were arranged in a completely randomised design with eight replicates. Control plants were treated with water. Plants were watered as often as necessary and fertilised with 50 g of diamonium phosphate.

The experiment was terminated eight weeks after soil infestation. Plants were gently uprooted from pots and roots washed free of soil. Determination of root damage was based on a scale of 0-10 where: - 0 = complete and healthy root system, no infestation, 1 = very small galls only detected upon close examination, 2 = small galls as in 1, but more numerous and easy to detect, 3 = numerous small galls, function of roots seriously affected, 4 = numerous small galls, some big galls, majority of galls still functioning, 5 = 25% of the root system severely galled and not functioning, 6 = 50% of root system severely galled and not functioning, 7 = 75% of root system severely galled and lost for production, 8 = no healthy roots, plant still green , 9 = the completely galled root system is rotting, plant was dying, 10 = plants and roots dead (Zech, 1971).

Egg mass index (EMI) was used to determine the ability of each cultivar to support nematode reproduction. Galled roots were washed free of soil and then dipped in 1% solution of Phloxine B to stain the egg masses. An egg mass index scale of 0-5, where 0= 0, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100, and 5 = >100 egg masses per root system, was used (Taylor and Sasser, 1978).

Effect of Meloidogyne incognita on the expression of bacterial wilt of potato. A greenhouse test to determine the effect of Meloidogyne incognita infection on the expression of bacterial wilt in potato cultivars with varying levels of resistance to bacterial wilt was conducted from August to October 1998. Three potato cultivars namely Asante CIP-381381.20 (very susceptible), B53 (moderately susceptible), and Kenya Dhamana CIP- 800228 (resistant) were used in this test. Certified tubers of these cultivars were sown in pots containing heat-sterilised soil: sand (2:1) mixture. Treatments included plants inoculated with (i) R. solanacearum without root wounding (severing) (ii) R. solanacearum with root wounding by the procedure described by Winstead and Kelman (1952) (iii) Meloidogyne incognita and R. solanacearum without root wounding (iv) M. incognita and R. solanacearum with root wounding. Controls included (a) plants, which were not inoculated with any pathogen but with wounded roots (b) plants which were not inoculated with any pathogen but with root wounding.
Nematode inoculum was extracted from galled tomato roots following the procedure described by Omwega et al. (1988). Galled tomato roots were obtained from two-month old tomato plants that were inoculated with M. incognita when they were two weeks old. The roots were washed free of soil and thereafter dipped in distilled water for about 1 hr to allow any other form of nematodes to move out into the water leaving behind M. incognita eggs. The roots were then dipped in sterile distilled water that was being aerated by an aquarium pump. The eggs hatched into juveniles, which moved into the water in about 5-10 days. Inoculum consisted of 6000 Meloidogyne J₂ suspended in 10 ml sterile distilled water. Inoculation was done by pippetting the inoculum into the soil in indentations made in the root zone of each plant two weeks after planting.

R. solanacearum inoculum consisted of a bacterial suspension containing 10⁹cfu/ml in sterile distilled water. Inoculation was done within two hours of inoculum preparation using the soil drenching and root-severing method described by Winstead and Kelman (1952). Treatments were arranged in a completely randomised design with three replicates. Plants were rated on a weekly basis for bacterial wilt severity up to 30 days post inoculation. A scale of 0-5 (from wilted leaves to death) where, 0 = no symptoms, 1 = 1 leaf wilted, 2 = 2 or 3 leaves wilted, 3 = all the leaves wilted except the top 2 or 3 leaves, 4 = all leaves wilted, 5 = plant dead, was used (Winstead and Kelman, 1952). The experiment was repeated to validate the results. Data were subjected to analysis of variance and means were separated using Fisher's protected least difference test at P=0.05.

RESULTS

Population density of Meloidogyne spp. in potato fields. Table 1 shows the second stage juveniles of Meloidogyne spp. in soil obtained from different altitude ranges. The correlation between altitude and population densities of Meloidogyne spp. juveniles were not significant (P>0.05). The mean population densities of Meloidogyne spp. juveniles in soil collected from Nyeri, Nyandarua and Meru districts are depicted in Table 2. The mean population of Meloidogyne juveniles in 200cm³ soil was 45 in Nyeri and Nyandarua districts and 31 in Meru district. Variations in juvenile populations of Meloidogyne spp. were significant (P<0.05) among divisions of the districts.

Reaction of potato cultivars to Meloidogyyne incognita. Differences in galling and egg mass indices were significantly (P=0.05) different among the potato cultivars, 60 days after soil infestation with 6000 eggs and juveniles of M. incognita (Table 3). None of the cultivars was immune to M. incognita. Galling index was highest (5.5) in cv. KP93739.26 and lowest (1.9) in cv. Nyayo. The susceptible control, tomato cv. Cal J, had the highest (7.6) galling index. With the exception of potato cv. KP93739.26, tuber infection was not observed.

Differences in nematode reproduction among the cultivars tested were highly significant (P < 0.01). All cultivars supported nematode reproduction with highest (5.0) egg mass index being recorded in cultivars Kerr's Pink, KP93739.26, Desiree (CIP-800048), KP92633.26, B53 and the susceptible control, tomato cv. Cal J. Nematode reproduction was lowest in cultivars Nyayo and Tigoni (CIP-381381.13) both of which had an EMI of 4.4. A positive and significant (r = 0.484; P=0.001) correlation existed between egg mass index and galling index.

Nematode infection caused reduction of plant height in all the potato cultivars screened (Table 3). Cultivar Rutuku CIP-720097 recorded the lowest (12.4%) reduction while the highest (50.6%) reduction was observed in cultivar Dutch Robjin. Differences in plant height between infected and uninfected plants were significant (P=0.05) in cultivars Desiree, K.Pink, K. Dhamana, Mauritius, Tigoni and Nyayo.

Effect of Meloidogyne incognita on development and severity of bacterial wilt of potato. Differences in bacterial wilt severity were significant (P<0.05) among the treatments for all the three cultivars used in this test (Table 4). Bacterial wilt severity was higher in plants inoculated with R. solanacearum and M. incognita than those inoculated with R. solanacearum alone.

Analysis of variance revealed significant (P<0.05) differences in bacterial wilt severity among the three cultivars. Plants whose roots were severed and inoculated with the two pathogens had the highest (3.4) overall bacterial wilt severity. Wilt severity was lower (3.2) but not significantly (P>0.05) different in plants inoculated with both pathogens without root severing. Wilt severity in plants inoculated with R. solanacearum without root-wounding was significantly (P=0.05) lower (1.1) than in plants inoculated with R. solanacearum and whose roots were severed (2.6).

Disease progress in the three potato cultivars is depicted in Table 5. For all the cultivars, significant (P=0.05) differences in bacterial wilt severity were observed among potato plants inoculated with R. solanacearum alone without root wounding and those inoculated with R. solanacearum and M. incognita throughout the seven weeks. Bacterial wilt severity was consistently higher in plants whose roots were severed. When plants inoculated with R. solanacearum alone were compared with those inoculated with R. solanacearum in combination with M. incognita, significant (P<0.05), differences in wilt severity were observed in each of the three cultivars throughout the assessment period (Table 5). Bacterial wilt development was faster and the severity consistently higher in plants inoculated with the two pathogens. Similarly, comparisons between plants inoculated with the two pathogens simultaneously with severed roots and those treated the same way but with unsevered roots indicated non-significant (P=0.05) differences in each cultivar and for every week (Table 5).

Bacterial wilt severity was higher and development faster in wounded plants inoculated with R. solancearum in combination with M. incognita. Likewise bacterial wilt severity was higher in potato plants inoculated with R. solanacearum with root-wounding than in plants inoculated with both R. solanacearum and M. incognita without root wounding.

DISCUSSION

The occurrence of Meloidogyne spp. juveniles in soil samples obtained from potato fields in Nyeri, Meru and Nyandarua districts supports a previous report that root-knot nematodes are commonly associated with potato in the tropics (Jatala and Bridge, 1990). Although temperature is known to influence all components of nematode life processes and host parasite relations (Noe and Sikora, 1990), the differences in root-knot nematode populations recovered from the potato fields could not be attributed to variation in temperature. The low populations of Meloidogyne spp. in Meru district was unexpected since the district experiences the highest temperature. However, apart from temperature, other factors such as moisture and aeration may have come into play in determining the population dynamics of Meloidogyne spp. (Sasser and Freckman, 1987).

Root galling, nematode reproduction and stunted growth were observed on all nematode-infected potato cultivars with no indications of immunity to M. incognita. Potato cultivars, however, were differentially stunted, confirming reports of differential varietal response to nematode infestation (Jatala and Bridge, 1990). A compatible reaction characterized by penetration and development of M. incognita larvae to maturity was observed in all the cultivars. Some cultivars such as KP93739.26, B53, and KP 92633.26 had high gall index and egg mass index but were not significantly stunted following nematode infection. Such cultivars may be tolerant to nematode infection.

Tubers obtained from nematode infected plants did not show galls or egg masses. According to Jatala (1975), tubers become infected when conditions are optimal. Penetration of M. incognita juveniles and symptom development in tubers require relatively higher temperatures (Charchar and Moita, 1996) than those required for parasitism of potato roots (Mai et al., 1981). Lack of galling on tubers, 60 days after inoculation in all the potato cultivars except KP 93739.26, may be attributed to the low temperatures that prevailed during the experiment. The mean atmospheric temperature at the time of the experiment was 17.2⁰C. Although no cultivar showed immunity to Meloidogyne, cultivars such as Nyayo, Tigoni, and Furaha can be recommended for soils heavily infested with Meloidogyne spp.

Although results from greenhouse screening may not be reproduced under field conditions (Janssen et al., 1996), it is conceivable that damage as a result of Meloidogyne infection in the field would be more severe because nematode infected roots are predisposed to other pathogens (Jatala and Bridge, 1990). In this evaluation, other soil-borne pathogens were destroyed through sterilization of the potting mixture. Effects of nematode damage, which are usually exaggerated by moisture stress, due to inability of roots to absorb moisture effectively, may have been reduced by regular watering of the plants.
Greenhouse studies revealed that infection by R. solanacearum in the presence of root-knot nematodes resulted in a higher severity and a faster development of bacterial wilt. Similar findings were reported by Ravichandra et al. (1990) and Akiew et al. (1991). The fact that bacterial wilt development was faster and severity consistently higher in plants inoculated with R. solanacearum in combination with Meloidogyne incognita than in plants inoculated with R. solanacearum alone suggests that the resistance mechanism(s) of potato plants may have been weakened in the presence of Meloidogyne. Nagesh et al. (1997) suggested a breakdown of resistance in tomato plants inoculated with R. solanacearum together with M. incognita.

Our results showed that bacterial wilt severity was lower in potato cv. B53 plants inoculated with R. solanacearum with root-wounding than in plants inoculated with R. solanacearum and M. incognita without root wounding. This shows that the role of M. incognita in bacterial wilt development is more than just providing avenues for the entry of bacterium. According to Napiere and Quimio (1980) and Samuel and Mathew (1983), the synergism between the two pathogens was mostly associated with wounds caused during penetration of larvae into roots. However, findings by Sitaramaiah and Sinha (1985) suggested that nematode induced stress was more important as a wilt-triggering factor than the wounding. Biochemical and physiological changes resulting from infection by root-knot nematodes may be responsible for the enhancement of wilt. According to Trudgill (1991), the biochemistry of a plant is considerably altered following nematode infection. These changes may either weaken the chemical resistance mechanisms of the plant or modify conditions in the infected potato tissue making the plant more suitable for bacterial colonization (Khan, 1993). This is in harmony with the suggestion by Jatala (1975) that infection by one pathogen may alter the response of a host to subsequent infection by another.

The result of this study reinforces the need to control root-knot in fields heavily infested with R. solanacearum in order to gain maximum benefit from the use of wilt resistant potato cultivars. Use of potato cultivars resistant to Meloidogyne are recommended as a component of integrated bacterial wilt disease management.

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