PLANT-PARASITIC NEMATODES ASSOCIATED WITH CABBAGE IN NYANDARUA AND EMBU DISTRICTS KENYA

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1 PLANT-PARASITIC NEMATODES ASSOCIATED WITH CABBAGE IN NYANDARUA AND EMBU DISTRICTS KENYA 1 Maina M.J., J.W. Waceke 2 and G.M. Kariuki 1 1 Department of Plant and Microbial Sciences, Kenyatta University, P.O Box Nairobi. 2 Department of Agricultural Sciences and Technology, Kenyatta University, P.O Box Nairobi. Abstract A survey was conducted to determine the occurrence of plant parasitic nematodes (PPN) associated with cabbage (Brassica oleracea L. var capitata) in Nyandarua and Embu districts of Kenya. A total of 60 soil and roots samples were collected from six agro-ecological zones (AEZs). Nematodes were extracted from 100 g of soil per sample using a modified Baermann extraction tray technique while nematodes inside the roots were extracted using modified maceration and filtration technique. Roots were rated for galling index using a scale of 0-5, then stained in phloxine B and in NaOCl-acid fuchsin for egg masses and presence of endoparasitic nematodes. A total of eighteen (18) genera of PPN belonging to ten (10) families of the order Tylenchida and Dorylaimida were identified from both soil and roots. The populations of various PPN differed significantly (p<0.05). Lesion nematodes (Pratylenchus spp.) were detected in 58% of the root samples, followed by spiral nematodes (27%) while Meloidogyne spp., were third with 23%. The root galling ranged from 1 to 2 on the galling index signifying some level of resistance. Lesion nematodes were also present in the soil at a high absolute frequency of occurrence of 87%; Helicotylenchus spp. had 82% and Meloidogyne spp. 42%. Other nematodes of economic importance included stunt nematode Tylenchorhynchus spp., stubby nematode Paratrichodorus spp. and Trichodorus spp. with 67, 57and 78% absolute frequency of occurrence respectively. Xiphinema spp. and Longidorus spp. were present in relatively lower frequency (28% and 20% respectively) in soil and only 8% and 5% in the roots. Filenchus spp., Coslenchus spp. and Tylenchus spp. were present at a high frequency. This study therefore concludes that cabbage is a potential host to PPN and particularly Pratylenchus spp. and its use as a rotation crop should be evaluated. Key words: Brassica oleracea var capitata, Helicotylenchus spp., Meloidogyne spp., Pratylenchus spp. Introduction White head cabbage (Brassica oleracea L. var. capitata) is widely grown in Kenya as a major source of food and income. It is a rich source of vitamins and minerals when used as vegetables by human and as fodder for animals in addition to having medicinal values (Fahey et al., Plant parasitic nematodes remain a major challenge in crop production especially in developing countries. They cause annual losses estimated at USD125 billion worldwide (Chitwood, 2003). Cabbage has been reported to host PPN (Potter and Olthof, 1993; Waceke, 2007). On the other hand, cabbage has been in use as a rotational crop in nematode management because it is regarded as poor host to RKN (Bello et al., 2004; Pattison et al., 2006). Presence of PPN associating with cabbage raises concern over its use as a rotation crop in nematode management. Persistent use of cabbage by farmers as a rotational crop might allow nematodes hosted by cabbage to increase beyond the economic threshold or leading to emergence of a serious PPN initially unknown to be important. There was need therefore for an extensive survey of PPN associating with cabbage with the aim of identifying, estimating population density and documenting the PPN genera that may have a significant impact on agriculture in Nyandarua and Embu. Materials and Methods Soil and root samples were taken from 60 cabbage growing fields distributed in six agro-ecological zones (AEZs) of Nyandarua and Embu districts as described by Jaetzold and Schimidt (1983). Five of these AEZs were in Nyandarua district, namely; UH1 (a) at Mutarakwa in North Kinangop, UH1 (b) at Melangine in Olkalou, UH2 at Ngano in Ol-joro-orok, UH3 at Magumu in South-Kinangop, and UH 4-near Lake Ol-bolossat in Ndaragwa, the sixth one was UM1 at Manyatta in Embu. A garden trowel was used to collect soil from the cabbage rhizosphere up to a depth of 20 cm. Ten points per every cabbage field (Young, 1990) were chosen randomly but with bias to areas in the farm with crops showing reduced vigour indicating presence of PPN. Areas that had roots of weeds or other crop species were avoided whenever possible to avoid nematodes from non-target group (Knight, 2001). Ten soil and root samples collected from each farm were mixed homogeneously to constitute a composite sample. Roots were place together with corresponding soil samples in a plastic bag, sealed and transported in cool boxes to the laboratory for nematode extraction. Nematode extraction 613

2 Soil and roots were separated in the laboratory, soil samples were thoroughly but gently mixed. One hundred grams of each composite soil sample were used for nematodes extraction by the modified Baermann funnel technique as described by Hooper et al., (2005). Nematodes obtained were enumerated using a dissecting microscope and the total population recorded as the number of nematodes 100 g of soil. The roots were washed free of soil and root gall severity was rated on a 0-5 scale: 0 no galls; 1= 1-2 galls; 2= 3-10 galls; 3= galls; 4= galls; and 5 more than 100 galls (Taylor & Sasser, 1978). Each root system was divided into three sub-samples; one of the sub-sample (10 g) was stained in phloxine B (Holbrook et al., 1983) for egg masses, another 10 g was cleared with NaOCl and stained in acid fuchsin to identify presence of endoparasitic nematodes which were then enumerated. Ten grams sub-sample per farm was taken for nematode extraction using the modified maceration and filtration technique, (Hooper et al, 2005) after maceration in a kitchen blender. Cysts forming nematodes were extracted from soil samples that had been air-dried in an open paper bag for more than two weeks. The cysts extraction and counting was done as described by Mennan and Handoo (2006). Plant parasitic nematodes were enumerated and identified to genus based on morphological characteristics, except free living nematodes which were counted but not characterised. The total nematode population was expressed as number of individuals per 100 g of dry soil and 10 g of roots. Specimens were killed in 60 C hot water, fixed in TAF and mounted in glycerol for identification using stereo microscope (LEICA DM 2500) at various magnifications. Data analysis Data collected was normalized using logarithmic transformation (Log 10 [x+1]) prior to analysis of variance. Means that were considered significantly different (P 0.05) were separated by LSD. Absolute frequency was determined using the following formula; Absolute frequency= 100 (Norton 1978). Results The population density of PPN and free living nematodes varied significantly (p<0.05) between agro-ecological zones (Figure 1). Fig. 3: Mean nematodes extracted from the rhizosphere of cabbage in Nyandarua and Embu districts Manyatta had the lowest PPN population density with mean of 28 nematodes/100 g of soil while the highest density was recorded in North-Kinangop which had a mean of 584 nematodes/100 g of soil. Free living nematodes were more abundant in South- Kinangop while Ndaragwa recorded the lowest. The number of soil PPN for the entire samples ranged from 12 to 584 individuals per 100 g dry soil. The highest population of root nematodes was recorded in Ol-kalou with a mean of 60 nematodes per 10 g of root while Manyatta had an average of less than 2 nematodes per 10 g of roots. Nineteen genera of PPN belonging to ten families in the order Tylenchida and Dorylaimida were found in association with cabbages rhizosphere and roots from 60 samples (Table 1). 614

3 Table 5: Genera of plant parasitic nematodes, means ± std error and absolute frequency (A/f) in Nyandarua and Embu districts Order Family Genus Soil samples Root samples Mean ± std A/f Mean A/f % error % Tylenchida Pratylenchidae Pratylenchus Filipjev 41.0±18.0 h ±8.4 b 58 Hoplolaimidae Helicotylenchus Steiner 26.3±8.3 gh ±0.3 a 27 Hoplolaimus Daday 0.5±0.1 a 13 Rotylenchulus Linford 1.6±0.6 a-c 27 & Oliveira Peltamigratus Sher 0.5±0.5 a 8 Scutellonema Andrassy 2.77±1.42 a-d ±0.8 a 10 Heteroderidae Heterodera Schmidt 5.7±2.1 b-e 33 Meloidogynidae Meloidogyne Goeldi 6.4±2.9 b-e ±0.8 a 23 Criconematidae Hemicycliophora 0.7±0.5 ab 15 de Man Tylenchidae Tylenchus Bastian 4.6±2.3 b-e 52 Filenchus Andrassy 16.2±6.1 f-h 88 Coslenchus Siddiqi 10.3±4.8 d-f 60 Tylenchorhynch idae Tylenchorhynchus Cobb 11.9±3.4 e-h ±0.2 a 22 Paratylenchidae Paratylenchus 6.2±3.0 c-f 58 Micoletzky Dorylaimida Longidoridae Longidorus Micoletzky 1.1±0.7 ab ±0.3 a 5 Xiphinema Cobb 2.0±1.4 a-c ±0.4 a 8 Trichodoridae Paratrichodorus 7.8±1.7 e-g 78 Siddiqi Trichodorus Cobb 6.7±3.4 c-e ±.1 a 1 Means within same column followed by the same superscript(s) are not significantly different (P<0.05) denote absence of the nematode genus Nematodes of the genus Pratylenchus spp. were detected in 58% of the root samples (Table 1), while 27% and 23% of the samples contained members of the genera Helicotylenchus spp. and Meloidogyne spp. respectively. Tylenchorhynchus spp. Scutellonema spp., Xiphinema spp., Longidorus spp. and Trichodorus spp. occurred less frequently in 22, 10, 8, 5 and 3% of cabbage roots respectively. Pratylenchus spp. occurred in 87% of the soil samples, 82% contained Helicotylenchus spp., 78% and 67% of the soil samples contained Paratrichodorus spp. and Tylenchorhynchus spp. respectively. Heterodera spp., Longidorus spp., Hoplolaimus spp. and Hemicycliophora spp. were less frequent occurring at 33, 20, 15 and 13% respectively. Filenchus spp., Coslenchus spp. and Tylenchus spp. occurred in high frequencies of 88%, 60% and 52% respectively. There was a significant difference at P<0.05 between various PPN genera as indicated in Table 1. Lesion (Pratylenchus spp.) was the most abundant nematode genera in cabbage roots with an average of 16 nematodes per 10 g of roots. It was significantly (P<0.05) higher than all other root nematodes as revealed by LSD post ANOVA analysis test. Meloidogyne spp., Scutellonema spp. and Helicotylenchus spp. had a mean population density of 1.1, 0.8, and 0.7 nematodes per 10 g of roots respectively; however, there was no significant difference between them (P>0.05). 615

4 A B C D Plate A: Galled cabbage roots stained in acid fuchsin from one of the farms in Ol-joro-rok Plate B: Helicotylenchus spp. ( 20) Plate C: Trichodorus spp. ( 20) Plate D: A mixed group of (mature male and female) Pratylenchus spp. ( 10) collected from a farm in Milangine; Ol-kalou. Pratylenchus spp., Helicotylenchus spp., Tylenchorhynchus spp. and Filenchus spp. were the most abundant in soil samples. They occurred in an average of 41, 26, 16 and 11 nematodes per 100 g of soil respectively. Root-knot nematodes (RKN) were present in low frequency in the roots (23%) scoring between 1 and 2 in the galling score. They occurred in 42% of the soil samples, with the highest frequency (70%) recorded in Ol-joroorok. Cysts were extracted from 82% of the soil samples (Plate F) the mean number of cysts per 100 g of soil ranged from 9 to 72, the highest number of cysts was recorded in a sample from Ol-joro-orok where 261 cysts were counted. 616

5 E F Plate E: Parasitic nematode inside cabbage root stained with acid fuchsin. Plate F: Cysts extracted from a farm in Ol-joro-orok. Discussion The results of this study show that cabbage is a host to economically important PPN which cut across major AEZs in Nyandarua and Embu with very slight difference in their population densities. With the exception of Peltamigratus spp. and Heterodera spp. which are being reported in cabbage rhizosphere in Kenya for the first time, all other nematodes herein reported have been reported infecting cabbage in Kenya (Waceke, 2007) and other parts on the world (Mennan and Handoo, 2006). The most frequent and abundant genera, represent the major PPN of cereal but have also been reported in vegetables (Netscher and Sikora, 1990). Some of the nematodes recovered in this survey, are considered to be seriously detrimental to vegetable production. Rotylenchulus spp. is known to affect vegetables though often neglected or overlooked where it occurs alongside Meloidogyne spp. (Netscher and Sikora, 1990). Pratylenchus spp. was widely spread and abundant, perhaps indicating it is among the major PPN nematodes of cabbage in Nyandarua and Embu. Waceke (2007) reported that it is well distributed in Kiambu and Kajiado districts of Kenya. Pratylenchus penetrans for instance has been reported to reduce market yield of cabbage by 19% to 33% when infested into cabbage grown in micro-plots (Olthof and Potter, 1973). Pratylenchus thornei was frequently distributed in Turkey (Mennan and Handoo, 2006) and P. zeae, P. scribneri, P. negrectus and P. loosi reported as present Kenya (Waceke, 2007). High presence of lesion nematode could be attributed to intercropping cabbage with other crops such as maize and oat as it was noted during the survey. The presence of lesion nematodes in cabbage roots could have serious implications regarding the use of this crop as a cover crops or rotational crop in cabbage and cereal production. Helicotylenchus spp. (spiral nematode) was the second most frequent and abundant nematodes in roots but third in soil samples. The spiral nematodes have a wide host range(kashaija, 1994) making them to survive in various cropping systems, their high numbers may have been contributed by farm practices such as crop rotation and intercropping in the areas. Helicotylenchus spp. has been reported in Kenya, Uganda and other parts of the world (Bafokuzara, 1996) associated with cabbage. Meloidogyne spp. occurred at a frequency of 23% in the roots, while the galling severity and egg mass indices were less than three in all farms sampled (Plate A). They occurred at relatively low density, though RKN are considered as potential pest of vegetables including cabbage (Netscher and Sikora, 1990; Bridge et al., 1996). Meloidogyne incognita has been reported as a pest of cabbage (Bridge et al., 1996; Mennan and Handoo, 2006). Presence of Meloidogyne spp. in cabbage roots and rhizosphere indicates their ability to infect cabbage, but the low frequency confirms cabbage as a poor host to root knot nematodes. This is in-line with previous findings with cabbage and crucifer group of crops (Waceke, 2007; Mennan and Handoo, 2006; Liébanas and Castillo, 2004) which are reported to possess some degree of resistance to RKN (Manfort et al., 2007). Detection of cysts in 82% of the samples indicates high presence of cyst nematode; Heterodera spp., although only 33% of Heterodera spp. juvenile were extracted from soil samples. A sample from Ol-joro-orok recorded the highest figure of cysts 261 per 100 g of soil. Cysts nematodes are known pest of cabbage in various parts of 617

6 the world (Mennan and Handoo, 2006). This could be the first record of cyst nematode associated with cabbage in Kenya. The importance of some genera identified in high frequency (Tylenchus spp., Filenchus spp. and Coslenchus spp.) as pathogens has not been established according to Hafez et al., (1992), Peltamigratus spp. has been reported in vegetables and is suspected to cause damage when in high population (Bridge et al., 1996). The presence of some important nematodes could be attributed to farming practices such as intercropping or continued cultivation of the same crop for several years as it emerged during the survey. Lack of use of nematodes control interventions, inadequate knowledge and technical knowhow by farmers, could also have contributed to high nematode densities. Conclusion and Recommendations Various surveys have indicated that nematodes are common pest of cabbage and are key constraints to vegetable production; the results discussed here have identified key nematodes. Pathogenecity studies are required to quantify damages caused by individual nematode genera. There is need for continued research in order to come up with cheap yet effective nematodes management techniques. This may include screening various cabbage species for resistance to various PPN with an aim of designing a proper nematode management strategy. Acknowledgement The first author is thankful to German Catholic Academic Exchange Program (KAAD) for funding this research. Thanks are also due to cabbage farmers in Nyandarua and Embu District for permitting collection of samples from their farms. References Bafokuzara, N.D. (1996). Incidence of Different Nematodes on Vegetable and Fruit Crops and Preliminary Assessment of Yield Loss Due to Meloidogyne Species in Uganda. Nematologia Brasileira, 20(1). Bello, A., Lopez, J.A., Garcia-alyarez, R.S. & Lacasa, A. (2004). Biofumigation and nematodes control in the mediterranean region. In: Nematology monographs and perspectives. Proceedings of the forth international congress of nematology 8th-13th June 2002 (pp. Pp ). Bridge, A., Hunt, D.J. and Hunt, P. (1996). Plant Parasitic Nematodes of crops in Belize. Nematropica. 26, Chitwood, D. (2003). Reseach on plant parasitic nematodes biology conducted by United States Department of Agriculture-Agriculture research services. Pest Management Science, 59, Fahey, J.W., Zalemann, A.T. and Talalay, P. (2001). The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry, 56, Hafez, S.L., Golden, A.M., Rashid, F. and Handoo, D. (1992). Plant parasitc nematodes associated with crops in Idayo Eastern Origon. Nematropica, Vol. 22, No 2. Holbrook, C.C., Knauft, D.A. and Dickson, D.W. (1983). A technique for screening peanut for resistance to Meloidogyne arenaria. Plant Disease, 67, Hooper, D.J., Hallmann, J. and Subbotin, S. (2005). Methods for extraction, processing and detection of plant and soil nematodes. In: Luc, M., Sikora, R.A. and Bridge (Eds.). Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, 2 nd edition. Wallingford, U.K: CAB International. Pp Jaetzold, R. and Schimidt, H. (1983). Farm management handbook of kenya Vol 11B and C. Ministry of Agriculture Nairobi, Kenya. Jenkins, W. (1964). A rapid centrifuge floatation technique for separating nematodes from soil. Plant Disease Report, 48: 692. Kashaija, I.N., Speijer, P.R., Gold, C.S. and Gowen, S.R. (1994). Occurrence, Distribution and Abundance of Plant Parasitic Nematodes of Banana in Uganda. African Crop Science Journal, 2 (1): Knight, K. (2001). Plant parasitic nematodes associated with six tropical crops in New Zealand. New Zealand Journey of Crop and Horticultural Science, 29: Liébanas, G. and Castillo, P. (2004). Host suitability of some crucifer for root knot nematodes in Southern Spain. Nematology, 6 (1)

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