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23Russian Journal of Nematology, 2023, 31 (1), 17 - 28
Prevalence, occurrence and characterisation of
disease complex involving Ralstonia solanacearum and Meloidogyne spp. on potato (Solanum tuberosum L.) in eastern Ethiopia
Tasew Getu1, Wassu Mohammed1, Awol Seid1, Tesfamariam Mekete2, Bekele Kassa3
and Mesfin Bogale4
'College of Agriculture and Environmental Sciences, Haramaya University, P.O. Box 138, Dire Dawa, Ethiopia 2United States Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine, National Identification Service, 4700 River Road, 20737, Riverdale, MD, USA 3Ethiopian Institute of Agricultural Research, Holetta Agricultural Research Center, P.O. Box 2003, Addis Ababa, Ethiopia ^Department of Entomology and Nematology, University of Florida, 32611, Gainesville, FL, USA
e-mail: [email protected]
Accepted for publication 4 May 2022
Summary. A survey was conducted to assess the prevalence, incidence and occurrence of Meloidogyne spp. and Ralstonia solanacearum (RS) disease complex on potatoes. A total 92 tubers and 92 soil samples were collected from 92 fields. Two species of root-knot nematodes, Meloidogyne incognita (MI) and M.javanica (MJ), and a bacterial isolate of RS were identified alone or in mixed populations. The highest prevalence of the Meloidogyne spp. and RS disease complex was 87% at Kersa. The highest incidence of the disease complex was 70% at Kersa and Chiro. In the tuber samples, the occurrence of the MI, MJ and RS disease complex achieved the highest mean value of 84.6% at Kersa. Farmers' awareness about these pathogens seems to be good. The study confirmed co-occurrence of the Meloidogyne spp. and RS in a single host and it indicates the need to examine the importance of these pathogens. Key words: bioassay, co-occurrence, incidence, Meloidogyne javanica, Meloidogyne incognita, root-knot nematode, survey.
Potato (Solanum tuberosum L.) is the world's third most important food crop after wheat and rice in terms of human consumption (FAOSTAT, 2019). It yields more food more rapidly than any other major crop, provides more nutrition than many other crops, and provides income for many small-scale farmers (Devaux et al., 2020). Unfortunately, the crop suffers from attacks by plant-parasitic nematodes, bacteria and other pathogens, which cause huge losses in production.
Root-knot nematodes (Meloidogyne species; RKN) are sedentary endoparasites that are highly polyphagous, global in distribution (Moens et al., 2009), and can cause up to 100% yield losses on tomato (Seid et al., 2015). Overall, plant-parasitic nematodes alone cause an estimated annual crop loss of $US 78 billion worldwide and an average crop yield loss of 10-15%. Nematodes alone can cause average yield losses in potato of up to 12% (Lima et al., 2018).
Prevalance is the proportion of individuals in a population that have a disease at a particular time, regardless of when they were infected, sometimes also called the point prevalence. It is a measure of the disease burden (Campbell & Madden, 1990). Incidence measures the number of new cases of a disease in a population during a given period of time. Occurrence is an instance of disease happening (Madden & Hughes, 1999). In Ethiopia, the prevalence of Meloidogyne spp. alone or in mixed populations has been reported on a small number of horticultural crops, such as tomato (Tefera & Hulluka, 2000; Mandefro & Mekete, 2002; Kassie, 2019; Miheret et al., 2019; Seid et al., 2019), hot pepper (Abegaz et al., 2019) and rose (Meressa et al., 2014). Seid et al. (2019) reported the prevalence and occurrence of four major Meloidogyne spp., Meloidogyne incognita, M. javanica, M. arenaria and M. hapla on tomato, using DNA and protein-based identification
© Russian Society of Nematologists, 2023; doi: 10.24412/0869-6918-2023-1-17-28
techniques. The occurrence of M. incognita and M. javanica has also been reported in tomato cultivation areas in eastern Ethiopia (Seid et al.,
2019). However, there is limited research information on the incidence, which refers to the rate at which the disease is manifested in a certain population (Hussain et al., 2012), prevalence and occurrence of Meloidogyne spp. in major potato growing areas of Ethiopia.
Ralstonia solanacearum (bacterial wilt) is one of the most destructive potato diseases (EPPO,
2020). It is reported as a serious production constraint in Ethiopia, Uganda, Kenya, Madagascar, Rwanda, Burundi, Nigeria and Cameroon (EPPO, 2014). Bacterial wilt in potato has been detected in many parts of Ethiopia (Stewart, 1956; Kassa, 1996; Gebremedhin et al., 2006; Henok et al., 2007; Abdurahman et al., 2017; Tessema et al., 2020). Yield losses of 30 to 100% due to R. solanacearum were reported in Kenya and Uganda (Kinyua et al., 2005).
Formation of galls on the root system, suppression of yields as well as overall stunting of the plant, are typical symptoms of Meloidogyne spp. (Moens et al., 2009). Wilting and yellowing of the leaves, discoloured tuber eyes, a milky-white sticky exudate or ooze are major symptoms of R. solanacearum (Agrios, 2005). The disease complex (synergistic interaction) between Meloidogyne spp. and R. solanacearum on various hosts is widely recognised (Martin & Nydegger, 1982; Ateka et al., 2001; Pavithra et al., 2014; Sundaresh et al., 2017; Getu et al., 2021). Experiments conducted in glasshouse conditions on various hosts revealed that the damage caused by concomitant infection by Meloidogyne spp. and R. solanacearum is more severe than that from separate infections (Sundaresh et al., 2017; Getu et al., 2021).
In eastern Ethiopia, research information on the prevalence, incidence and occurrence of Meloidogyne spp. and R. solanacearum, either alone or as a disease complex on potato, is limited. However, both Meloidogyne spp. and R. solanacearum are important and increasing constraints to potato cultivation in the region. Therefore, co-occurrence of Meloidogyne spp. and R. solanacearum may be one of the most important factors responsible for potato yield reduction. The main objective of the present research was to study the prevalence, incidence and occurrence of Meloidogyne spp. and R. solanacearum disease complex on potato; and to assess the farmers' perception and management practices against these diseases in the main potato growing districts of eastern Ethiopia.
MATERIAL AND METHODS
Description of the study areas. Six districts namely, Haramaya, Kersa, Meta, Kombolcha and Jarso from the east Hararghe Zone and Chiro from west Hararghe Zone of eastern Ethiopia, were included in this survey. The survey was conducted between the 2nd and 4th week of September 2018. In addition, Haramaya University Raree Research Station (HURRS) were included in the survey.
Root and soil sample collection. Tuber and soil samples were collected from the selected potato growing districts of eastern Ethiopia. Potato growing districts were selected after discussion with regional agricultural development agents on the potential of the areas for producing potato. From each district, three potato-growing villages and five farms in each village were surveyed. A total of 184 composite samples from 90 farms and two research stations (92 soil and 92 root or tuber samples) were collected.
Tuber and soil samples were collected aseptically in a zigzag 'Z' sampling pattern, which considers pathogens distribution and nature of the field (Coyne et al., 2018). Samples were from 25 plants per farm, separately so that each tuber sample corresponded with a soil sample. Approximately 1.5 kg tubers per sample were collected. For soil samples, 30 soil core samples were taken from the top 25 cm depth using a 2 cm diam. auger next to the associated plant to make a bulk of about 2 kg soil (Hussey & Barker, 1973). The collected samples were bagged separately in polyethylene bags, tied with a twin thread, and then labelled with necessary field information, and placed immediately inside the ice-box at approximately 5°C for preservation (Coyne et al., 2018). Potato fields about 3 km apart from each other were selected with the help of the regional development agents. During sample collection, all possible care was taken to avoid cross-contamination. The collected samples were transported to Haramaya University and stored in a refrigerator at +5°C to keep the samples fresh (Coyne et al., 2018). The laboratory work was performed in the Postgraduate Plant Pathology Laboratory of Haramaya University, Ethiopia, but the molecular identification of the Meloidogyne spp. was carried out at the Nematode Systematics and Biological Control, University of Florida, USA.
For the field survey data collection purpose, semi-structured questionnaires were prepared and provided to all potato growers from whom the samples were collected to assess the farmers' management practices and awareness of Meloidogyne spp. and R. solanacearum diseases on
potato. Selected interviews and focus group discussions were also made to extract more information about the farmers' practices and awareness of these diseases.
Bioassay test. Soil samples collected from each field were tested to detect the occurrence of Meloidogyne spp. and R. solanacearum disease complex with a bioassay test using the susceptible tomato 'Marmande' as an indicator plant. Three-week-old tomato seedlings or seedlings that reached the four leaf-stage were grown on all collected soil samples in 500 cm3 size pots under glasshouse conditions. After 8 weeks, the root system from each pot was collected and was diagnosed for Meloidogyne spp. and R. solanacearum infection separately.
Nematode extraction. Root-knot females were
excised from tuber and tomato root galls using a sterilised scalpel, needle and a stereoscopic microscope. Females dislodged from each tuber and root samples were grouped for each sample in a vial containing water for identification.
Identification of Meloidogyne spp. Identification of the species of Meloidogyne collected from potato fields was done based on perennial patterns morphology (Taylor & Sasser, 1978; Hunt & Handoo, 2009) and using BLAStn analysis of the primer MORF (ATC GGG GTT TAA TAA TGG G) / MTHIS (AAA TTC AAT TGA AAT TAA TAG C) sequences (Pagan et al., 2015).
Ralstonia solanacearum isolation and purification. Ralstonia solanacearum isolation was done from diseased potato tubers showing symptom milky white bacterial cell ooze (Shew & Lucas, 1991) and without clear symptoms, and from the roots of tomato (bioassay test). The samples were macerated, washed with tap water, surface-sterilised in 70% ethanol for 3 min, rinsed in sterilised distilled water and then dried with tissue paper (EPPO, 1990). About 5 g lesions of tuber or root of tomato from each sample were cut aseptically and diseased tissue placed in sterile distilled water, separately. The samples were left to stand for 10 min to allow any bacteria present to be released into a sterile Petri dish containing sterile distilled water; 0.1 ml of the macerate was dispersed on the surface of nutrient agar (NA medium; beef extract 3.0 g, agar 15.0 g, peptone 5.0 g l-1) with a sterile loop and incubated at 28°C for 48 h (Goszczynska et al., 2000).
Presumptive R. solanacearum colonies were purified by streaking a single colony of each isolate on 2, 3 and 5-triphenyltetrazolium chloride (TTC solid medium; peptone 10.0 g, casein hydrolysis 1.0 g, glycerol 5 ml, and agar 15.0 g l-1). All the media were sterilised by autoclaving at 121°C for 15 min.
In TTC medium, 5 ml of 1%, 2, 3, 5 TTC was added to the sterilised 1 l medium before pouring. It was incubated at 28°C for 48 h (Kelman, 1954).
Ralstonia solanacearum identification and culture maintenance. To identify the bacterial species, the isolates were subjected to morphological, physiological and biochemical tests. Morphological identification was done according to Kelman (1954).
Gram staining. For the Gram staining test, a young 48 h-old bacterial colony was tested to identify whether the isolates were Gram-positive or Gram-negative bacteria. All specimens were examined under an oil immersion microscope (Gregerson, 1978).
KOH solubility test. To see if a mucoid thread can be lifted with the loop or not, a drop, approximately 0.25 ml, of potassium hydroxide (KOH) (3% aq., w/v) was placed using a Pasteur pipette on a clean glass slide (Ryu, 1940). Bacterial cells were transferred from a young culture media aseptically with a loop and placed into the drop of KOH with rapid, circular agitation. After 5-8 sec, the loop was alternatively raised and lowered just off the slide surface to detect a stringing effect. The KOH test was considered positive if drop viscosity or mucoid increased and stringing occurred within 15 s (Suslow et al., 1982).
Oxidative use of carbohydrates. Two test tubes containing basal mineral medium plus 5 ml 10% sucrose solution (w/v) were stab-inoculated with bacteria taken from a young colony. Two drops of sterile mineral oil were added to one tube then the tubes were incubated for 3 weeks to see oxidative reaction (Goszczynska et al., 2000).
Levan production. Levan is a homopolysaccharide of fructose units that is produced by some plants and microorganisms (Gonzalez-Garcinuno et al., 2017). Test tubes containing NA with 5% sucrose were streak-inoculated with young colonies and incubated at 28°C for 5 days. Levan (convex, white, domed and mucoid colonies) production was tested (Schaad, 1980). It was evaluated through observing convex, white, domed and mucoid colonies (Goszczynska et al., 2000).
Decomposition of nitrogenous compounds or arginine dihydrolase. Test tubes containing arginine medium were stab-inoculated with a young bacterial colony then covered with sterile mineral oil and incubated at 28°C. After 48 h the tubes were checked to determine if changes from yellow to pink colour occurred (Goszczynska et al., 2000).
Starch hydrolysis. Starch plates were streak-inoculated with a young 24 h-old bacterial colony,
which was the same for all tests per sample, and incubated for 1 week then flooded with iodine solution (Goszczynska et al., 2000).
Catalase. A few drops of 3% H2O2 were added to a 24 h-old bacterial colony on a plate to test for gas bubble formation (Goszczynska et al., 2000).
Biovar tests. To the olive-green coloured basal medium, 10% solution of hexose alcohols (inositol, mannitol, sorbitol) and carbohydrates (cellobiose, sucrose, maltose) were added in 5 ml amounts into sterilised test tubes (150 mm x 10 mm) in 20 ml amounts to group all bacterial isolates into different biovars based on the colour changes. From an already prepared 2-day-old bacterial suspension culture, 0.1 ml was added to two test tubes (control with no carbohydrate). The tubes were incubated at 30°C then examined at 3, 7 and 14 days (Hayward, 1964).
Farmers' awareness and practices towards Meloidogyne spp. and Ralstonia solanacearum. A cross-sectional analysis study was conducted to assess farmers' awareness and management practices towards Meloidogyne spp. and R. solanacearum. In the study, a total of 180 respondents (through questionnaire and interview) were incorporated. The independent variables used in the survey were previous crop, irrigation practice, management practice, pesticide used, fertiliser used, source of seed, and level of awareness that potato growers have about Meloidogyne spp. and R. solanacearum diseases and/ or disease complex. The dependent variables used in the survey were to quantifty Meloidogyne spp. and R. solanacearum damage symptoms by showing the photos of damaged plants.
Data analysis. The prevalence of Meloidogyne spp. and R. solanacearum disease complex were assessed after identification at the laboratory by the formula as a total number of fields with disease complex / total number of fields surveyed x 100, while the incidence was determined by the total number of infected plants with disease complex / total number of observed plants x 100 (Hussain et al., 2012). The occurrences of species were computed by number of samples with species / total number of samples observed x 100 (Norton, 1978). The data obtained from the survey and glasshouse (bioassay) were subjected to analysis of variance using SAS software version 9.2 (SAS Institute, 2009) and data of a cross-sectional study were analysed using SPSS version 20 software (SPSS, 2020). The binary logistic regression analysis (odds ratio with corresponding 95% confidence interval) was performed to assess the presence and degree of
association between the dependent and independent variables. To observe the significant differences, the mean value of the data was compared using Duncan Multiple Range Test (DMRT) at P < 0.05 (Gomez & Gomez, 1984).
RESULTS
Identification of Meloidogyne spp. The identification of root-knot nematodes using morphological characters of perineal patterns of females extracted from tubers collected from surveyed areas and bioassay tests demonstrated the presence of M. incognita and M. javanica. The important distinguishing features of perineal patterns ofM. incognita were typically high with a square-like dorsal arch. Striae were distinct and wavy. The dorsal striae were smooth, closely placed wavy to zigzag; the dorsal and ventral striae appearing to be interrupted and forked at the lateral line. Meloidogyne javanica perineal patterns had a low and rounded dorsal arch, lateral ridges that divide the pattern into dorsal and ventral regions or striae. Striae were coarse and smooth to slightly wavy and tail terminus often with distinct curl. Meloidogyne javanica was different from other Meloidogyne spp. by containing its pattern on the lateral field (Taylor & Sasser, 1978; Hunt & Handoo, 2009). In addition, BLAStn analysis of the MORF/MTHIS sequences (Pagan et al., 2015) showed the tested isolates were all M. incognita. Although only 30 (out of 90) samples were identified molecularly, all results supported the identification of Meloidogyne spp. by morphological characteristics.
Ralstonia solanacearum identification. Morphological. All R. solanacearum isolates produced cream-coloured bacterial colonies on nutrient agar (NA) media after 24 h incubation at 28°C. All isolates produced drop-shaped white colonies with pink, mucoid, or half-moon shapes on TTC media after 48 h incubation at 28°C as described by Kelman (1954).
Physiological. The result of the Gram staining test indicated that all bacterial isolates were Gramnegative (red) (Gregerson, 1978). A mucoid-like viscous thread was lifted from bacterial colony with the help of a loop, and the solution stuck to the loop when touched. This confirmed that the isolates were Gram-negative bacteria (KOH solubility test) (Suslow et al., 1982). A colour change to yellow was observed only in open (without mineral oil) tubes, which confirmed the oxidation reaction; the isolates were identified as R. solanaceurum (oxidative use of carbohydrates) (Goszczynska et al., 2000).
Fig. 1. Prevalence and incidence (%) of Meloidogyne spp. and Ralstonia solanacearum disease complex in the surveyed areas of eastern Ethiopia. The bar shows standard error at P < 0.05. HURRS = Haramaya University Raree Research Station.
.a 100
■u
(J
JJ
■1)
'-J £
— Pn
so 60 40 20 0
93 5
III
PMS IMS PRS iRS PMSRS IMSRSi Pathogens
Fig. 2. Overall prevalence and incidence of Meloidogyne spp. and Ralstonia solanacearum and / or disease complex in the surveyed areas of eastern Ethiopia. PMS = prevalence of Meloidogyne spp. (MS), IMS= incidence of MS, PRS = prevalence of R. solanacearum (RS), IRS= incidence of RS, PMSRS = prevalence of MS and RS, IMSRS = incidence of MS and RS. The bar shows standard error at P < 0.05.
Biochemical. Levan production test has been shown that convex, white, domed and mucoid colonies were not produced from sucrose, which shows that the isolates were R. solanacearum (Schaad, 1980). After 48 h the tube contents changed from yellow to pink, which confirmed that the bacterial isolates were R. solanacearum (decomposition of nitrogenous compounds (arginine dihydrolase)) (Goszczynska et al., 2000). The starch hydrolysis test also showed a blue-black, clear zone
around bacterial growth indicating starch hydrolysis (amylase activity) (Goszczynska et al., 2000). The result of the catalase test (formation of a gas bubble) further indicated that the isolates were R. solanacearum (Goszczynska et al., 2000). The results with all bacterial isolates indicated that acid pH (yellow colour) was observed from the top downwards of the test tubes between 3 to 5 days. This confirmed the isolates to be biovar II (Hayward, 1964).
Prevalence and incidence of Meloidogyne spp. and Ralstonia solanacearum disease complex. Out
of the 92 tuber and 92 soil samples tested in the bioassay, 78 (85%) and 63 (68.5%) were found to be infested by Meloidogyne spp. and R. solanacearum, respectively. The prevalence of the Meloidogyne spp. and R. solanacearum disease complex varied between the surveyed districts with the highest prevalence, 87%, at Kersa as opposed to the 50% at HURRS. In the surveyed areas, the incidence of the disease complex was also variable, with the highest 70% at Kersa and Chiro, and the lowest at Jarso 42% (Fig. 1).
Overall, the prevalence and incidence of the Meloidogyne spp. and R. solanacearum disease complex in the surveyed areas of eastern Ethiopia were found to be 70% and 58%, respectively. Regardless of disease complex, prevalence of the Meloidogyne spp. (93.5%) and R. solanacearum (79%) and incidence of the Meloidogyne spp. (59%) and R. solanacearum (70.4%) were recorded (Fig. 2).
Occurrence of Meloidogyne spp. and Ralstonia solanacearum disease complex in the tuber and soil samples. Meloidogyne spp. and R. solanacearum disease complex: M. incognita and R. solanacearum (MIRS), M. javanica and
R. solanacearum (MJRS), M. incognita, M. javanica and R. solanacearum (MIMJRS) were observed, and varied in the surveyed areas. In the tuber samples, the occurrence of the MIMJRS achieved the highest mean value 34 (84.6%) at Kersa and the lowest 25 (61.6%) at Jarso. The MIRS achieved the highest mean value 29 (72.5%) at Kersa and the lowest of 21 (52.5%) at Jarso. In the soil samples (bioassay test), the occurrence of the MIMJRS achieved the highest mean value 32 (79.1%) at Kersa and the lowest 14 (35.0%) at HURRS. The MIRS achieved the highest mean values 27 (67.5%) at Kersa and the lowest 13 (31.2%) at Jarso. The occurrence of the Meloidogyne spp. and R. solanacearum disease complex in tubers was found to be higher than in the soil samples (Table 1). Tubers infected by Meloidogyne spp. and R. solanacearum diseases complex are shown in Figure 3.
Farmers' awareness of and practices against Meloidogyne spp. and Ralstonia solanacearum.
Most of the independent variables, previous crop (Solanaceous), sources of seed (farm saved and research centre), fertiliser (inorganic), farmers' awareness, factors for disease development, and control methods (fungicide, ploughing and crop rotation) had highly significant (p < 0.01) effect on
Table 1. Altitude range, occurrence of Meloidogyne incognita, M. javanica and Ralstonia solanacearum (MIMJRS), M. javanica and R. solanacearum (MJRS) and M. incognita and R. solanacearum (MIRS) disease complex in tuber and soil samples (bioassay test), collected from eastern Ethiopia with total number of fields surveyed per district.
Tuber Soil
District Altitude range (m a.s.l.) MIRS MJRS MIMJRS MIRS MJRS MIMJRS
(%) (%) (%) (%) (%) (%)
Kersa 1942-2075 72.5a(29) 27.5b(11) 84.6a(34) 67.50a(27) 25.8b(10) 79.1a(32)
Haramaya 1991-2115 58.3ab(23) 35.0a(14) 70.0b(28) 54.3bc(22) 32.3a(13) 65.1b(26)
HURRS 2008-2013 65.0b(26) 35.0a(14) 72.5ab(29) 31.2d(13) 18.7c(8) 35.0c(14)
Metta 2013-2131 60.5ab(24) 35.1a(14) 72.1ab(29) 55.3bc(22) 33.6a(13) 66.1b(26)
Chiro 2028-2442 64.3b(26) 35.6a(14) 76.5ab(31) 60.0ab(24) 33.3a(13) 71.3ab(29)
Kombolcha 2084-2197 53.8bc(21) 32.8ab(13) 64.8bc(26) 49.8c(20) 30.1ab(12) 60.0bc(24)
Jarso 2440-2747 52.5bc(21) 27.5b(11) 61.6c(25) 47.6c(19) 25.6b(10) 56.0bc(22)
Mean 60.9(24) 32.64(13) 71.73(29) 52.24(21) 28.49(11) 61.80(25)
Mean values not sharing common letter(s) within columns of each parameter differ significantly at P < 0.05 according to Duncan Multiple Range Test (DMRT). HURRS = Haramaya University Raree Research Station. Numbers in brackets are mean number of Meloidogyne spp. collected and identified from each fields of surveyed districts. m a.s.l. = metres above sea level.
the dependent variables. The highest effect on the dependent variable Meloidogyne spp. and R. solanacearum damage symptoms on potato tuber was from the fertiliser (Table 2). When shown symptoms of Meloidogyne spp. and R. solanacearum, 163 (90.5%) out of 180 respondents reported damage symptoms such as gall and wilting;
the remaing 17 (9.5%) did not report the damage symptoms. Out of the 180 respondents, 133 (74%) and 47 (26%) replied that their field in the previous season was cropped with solanaceous and non-solanaceous crops, respectively. Out of the 180 respondents, 164 (91%), 16 (9%) indicated that in the previous season the solanaceous and non-
solanaceous crops were grown with irrigation and rainwater, respectively. In those samples collected from where the previous crop was irrigated and in sandy soil type, higher Meloidogyne spp. related symptoms were observed (data not presented). The
sources of the seeds that respondents used to plant were examined. Out of 180 respondents, seed sources were 103 (57%) farm-saved, 65 (36%) obtained from a research centre, and 12 (6.6%) collected in local markets.
Table 2. Binary logistic regression analysis between the dependant variable (Meloidogyne spp. and Ralstonia solanacearum damage symptoms) and seven independent variables based on the odds ratio at P < 0.05 significance level.
Independent variables Meloidogyne spp. and Ralstonia solanacearum damage symptoms
163 (90.5%) 17 (9.5%) Odd ratio 95% confidence interval P value
Previous crop:
Solanaceous 133 (74%) 0.63 0.51-0.80 < 0.01
Non- solanaceous 47 (26%) 0.87 0.81-0.91 = 0.05
Sources of seed:
Farm saved 103 (57%) 0.77 0.70-0.88 < 0.01
Research centre 65 (36%) 0.85 0.79-0.92 < 0.01
Local market 12 (6.6%) 0.90 0.85-0.95 > 0.05
Sources of water:
Irrigation 164 (91%) 164 23-1157 < 0.01
Rain 16 (9%) 0.89 0.85-0.94 > 0.05
Fertilizer:
Inorganic 165 (92%) 82.5 20.8-327 < 0.01
Organic 15 (8%) 0.89 0.85-0.94 > 0.05
Awareness:
Meloidogyne spp. 132 (73%) 0.64 0.52-0.79 < 0.01
Ralstonia solanacearum 94 (52%) 0.80 0.72-0.89 < 0.01
Factors to disease development:
Climate change 135 (75%) 0.62 0.49-0.78 < 0.01
Pathogens 45 (25%) 0.87 0.82-0.93 < 0.01
Controlling methods used:
Fungicide 141 (86%) 0.59 0.46-0.76 < 0.01
Insecticide 10 (6.4%) 0.90 0.85-0.94 > 0.05
Herbicide 4 (2.5%) 0.90 0.86-0.94 > 0.05
Ploughing 132 (73%) 0.64 0.52-0.79 < 0.01
Crop rotation 117 (65%) 0.73 0.63-0.84 < 0.01
Host plant eradication 22 (12%) 0.89 0.84-0.94 > 0.05
Fallowing 3 (2%) 0.90 0.86-0.94 > 0.05
Fig. 3. Tuber infected by Meloidogyne spp. and Ralstonia solanacearum disease complex: A) tuber galling; B) transverse section of galled tuber infected with R. solanacearum.
In surveyed areas, farmers were practicing various management strategies towards Meloidogyne spp. and R. solanacearum. Out of 180 respondents, 155 (86%) used chemicals, out of 155 respondents, 141 (91%) applied fungicide, 10 (6.4%) applied insecticide, and 4 (2.5%) applied herbicide. Amazingly, none of the respondents used and/or were aware of nematicides and bactericides. Other management approaches were through cultural practices. All respondents (100%) practiced weeding with rather good success across the surveyed areas. Ploughing 132 (73%), crop rotation 117 (65%), host/infected plant eradication 22 (12%), fallowing 3 (2%), and combinations of two or more approaches or integrated disease management 166 (92%) were the remaining strategies.
DISCUSSION
Results of the identification of Meloidogyne spp. were based on the morphological characters of perineal patterns of females extracted from infected tubers and soil bioassay tests on soil collected from all surveyed areas and showed the species were M. incognita and M. javanica. Although only a few samples were identified molecularly, all results supported the identification of Meloidogyne spp. by morphological method.
The results revealed that the majority (> 68%) of the samples from soil and tubers were infested with both Meloidogyne spp. and R. solanacearum. The prevalence and incidence of the two pathogens suggest their importance as a potential threat to potato production in the surveyed areas; thus, management of these pests should be given due attention. The distribution of the diseases and the two disease complexes were uneven and it was highest in Kersa district (1942-2075 m a.s.l). The highest prevalence and incidence of the diseases in the Kersa district might be due to intensive vegetable cultivation and the elevations of the district lower in altitude than other districts. Seid et al. (2019) reported that nematode diversity is greater at lower altitudes than higher altitudes. Other reasons for the variation are probably due to factors related to differences in edaphic and climatic factors of the surveyed areas, quality of seeds, or planting materials that were from susceptible potato cultivars. The agro-climatic conditions of the areas, soil type and moisture are reported to affect the population of such soil-borne pathogens (Wallace, 1969; Davide, 1985; Sasser & Carter, 1985; Waals et al., 2013; Ami et al., 2018). Reports indicate that infection of Meloidogyne spp. increases with the increase in sand content (Lawrence et al., 1997;
Tefera & Hulluka, 2000; Karssen et al., 2013; Kayani et al., 2013; Zia et al., 2014). Seid et al. (2019) also reported greater Meloidogyne spp. damage in sandy soil type in tomato fields.
The prevalence and incidence of Meloidogyne spp. and R. solanacearum diseases complex on potato have not been reported in Ethiopia, yet. However, the prevalence of Meloidogyne spp. and Fusarium wilt (Fusarium oxysporum) on tomato crops have been reported (Kassie, 2019). Our study revealed the cooccurrence of the two pathogens in both a single host and soil, and synergistic interaction between the two pathogens in eastern Ethiopia. The synergistic interaction between Meloidogyne spp. and R. solanacearum on a variety of hosts, such as brinjal (Reddy et al., 1979; Pavithra et al., 2014), tamarillo (Martin & Nydegger, 1982), potato (Suatmadji, 1986; Ateka et al., 2001; Getu et al., 2021), tomato (Sundaresh et al., 2017), has been reported previously. Information on the co-occurrence of the two pathogens in all of the surveyed districts revealed the importance of the pathogens on potato. More research and development work are required to counteract the pathogens.
In line with the present work, the occurrence of the two predominant tropical Meloidogyne spp., M. incognita and M. javanica, has earlier been reported on tomato in Ethiopia (Tefera & Hulluka, 2000; Mandefro & Mekete, 2002; Kassie, 2019; Miheret et al., 2019; Seid et al., 2019).
The majority of the respondents (64%) used farm-saved seed tubers and seed tubers bought from local markets for planting. The use of non-certified seed might contribute towards the prevalence of Meloidogyne spp. and R. solanacearum disease complex in the surveyed areas. Certified planting materials that are clean and free from diseases are necessary for the management of the diseases. Mulatu et al. (2005) reported that most farmers of eastern Ethiopia grew local potato cultivars, except for some farmers in the vicinity of Haramaya University and farmers who are targeted by NGO seed programmes that have access to improved varieties. Hirpa et al. (2010) reported the problems of the seed systems in Ethiopia, which seem to provide seed tubers of insufficient quality, and the unavailability of seed tubers of improved varieties. The majority (86%) of the respondants applied chemicals such as fungicide, insecticide and herbicide in order to manage their fields. However, none of the respondents used and/or were aware of nematicides and bactericides. This might be due to unavailability of the chemicals in the nearby areas. However, the chemicals are registered for use in the country (Mengistie, 2016).
ACKNOWLEDGEMENTS
This work is part of a Ph.D. dissertation of the first author. The authors are thankful to the Ministry of Science and Higher Education (Mo SHE) and Haramaya University (HU) for their extended material, technical and financial support in carrying out this study.
REFERENCES
Abbot, W.S. 1925. Methods for computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267. DOI: 10.1093/jee/18.2.265a Abdurahman, A., Griffin, D. Elphinstone, J. Struik, P.C. Schulz, S. & Sharma, K. 2017. Molecular characterization of Ralstonia solanacearum strains from Ethiopia and tracing potential source of bacterial wilt disease outbreak in seed potatoes. Plant Pathology 66: 826-834. Abegaz, B., Seid, A., Meressa, H.B., Fininsa, C., Dejene, M. & Woldetsadik, K. 2019. Evaluation of hot pepper (Capsicum annum and C. frutescenes) genotypes for resistance and hatch stimulation to Meloidogyne incognita and M. javanica from Ethiopia. Russian Journal of Nematology 27: 83-96. DOI: 10.24411/0869-6918-2019-10009 Agrios, G.N. 2005. Plant Pathology. USA, Elsevier
Academic Press. 922 pp. Ami, S.N., Ghaeib, S. & Shingaly, A. 2018. Disease incidence, identification, and monthly fluctuations in the population density of root-knot nematodes Meloidogyne javanica on cucumber plants in Semel District, Duhok, Kurdistan Region, Iraq. Agriculture and Environment 10: 52-65. DOI: 10.2478/ausae-2018-0005 Ateka, E.M., Mwang'ombe, A.W. & Kimenju, J.W. 2001. Studies on the interaction Ralstonia solanacearum and Meloidogyne spp. in potato. African Crop Science Journal 9: 527-535. Campbell, C.L. & Madden, L.V. 1990. Introduction to Plant Disease Epidemiology. USA, John Wiley and Sons Inc. 532 pp. Coyne, D.L., Nicol, J.M. & Claudius-Cole, B. 2018. Practical Pant Nematology: A Field and Laboratory Guide. Nigeria, International Insitute of Tropical Agricultre (IITA). 96 pp. Davide, R.G. 1985. Summary report on the current status, progress and needs for Meloidogyne research in Region VI. In: An Advanced Treatise on Meloidogyne. Volume 1: Biology and Control (J.N. Sasser & C.C. Carter Eds). pp. 369-372. Raleigh (NC), USA, North Carolina State University / the U.S Agency for International Development.
Devaux, A., Goffart, J.P., Petsakos, A., Kromann, P., Gatto, M., Okello, J., Suarez, V. & Hareau, G. 2020. Global food security, contributions from sustainable potato agri-food systems. In: The Potato Crop: Its Agricultural, Nutritional and Social Contribution to Humankind (H. Campos & O. Ortiz Eds). pp. 3-35. Lima, Peru, International Potato Center. DOI: 10.1007/978-3-030-28683-5_1 EPPO. 1990. Standards PM 3/26 phytosanitary procedures: Ralstonia solanacearum. Inspection and test methods. EPPO Bulletin 255: 20-62. EPPO. 2014. Plant Quarantine Data Retrieval System Database (URL: https://www.eppo.int/RESOURCES/ (accessed: December 14, 2018)). EPPO. 2020. Global Database (URL: https://www.gd.eppo.int/taxon/RALSO (accessed: June 10, 2019)). FAOSTAT. 2019. Food Balance Sheet (URL: https://www.potatopro.com/africa/potato-statistics (accessed: October 5, 2019)). Gebremedhin, W., Solomon, A., Gebre, E., Tesfaye, K., Kassa, B.B, Lemma, D.Y. & Bedane, K. 2006. Transforming the traditional potato production through improved technologies in the central highlands of Ethiopia. In: Proceedings of the International Conference "Scaling up and Scaling out Agricultural Technologies in Ethiopia" (T. Abate Ed.). pp. 159-168. Addis Ababa, Ethiopia, Ethiopian Institute of Agricultural Research. Getu, T., Mohammed, W., Seid, A. Mekete, T. & Kassa, B. 2021. Reaction of selected potato cultivars to Meloidogyne incognita and Ralstonia solanacearum under greenhouse conditions. Archives of Phytopathology and Plant Protection 54: 14561470. DOI: 10.1080/03235408.2021.1914357 Gomez, K.A. & Gomez, A.A. 1984. Statistical Procedures for Agricultural Research. USA, John Wiley and Sons Inc. 680 pp. GonzAlez-Garcinuno, A., Tabernero, A., SAnchez-Alvarez, J., Miguel, A.G., Eva, M. & Valle, M.D. 2017. Effect of bacteria type and sucrose concentration on levan yield and its molecular weight. Microbial Cell Factories 16: 91. DOI: 10.1186/s12934-017-0703-z Goszczynska, T., Serfontein, J.J. & Serfontein, S. 2000. Introduction to Practical Phytobacteriology: A Manual for Phytobacteriology. South Africa, SAFRINET. 83 pp. Gregerson, T. 1978. Rapid method for distinction of gram-negative from gram positive bacteria. European Applied Microbialogy and Biotechnology 5: 123-127. Hayward, A.C. 1964. Characteristics of Pseudomonas solanacearum. Journal of Applied Bacteriology 27: 265-277.
Henok, K., Fassil, A. & Yaynu, H. 2007. Evaluation of Ethiopian isolates of Pseudomonas fluorescens as biological agent against potato bacterial wilt caused by Ralstonia (Pseudomonas) solanacearum. Acta Agriculturae Slovenica 90: 125-135.
Hirpa, A. Meuwissen, M.P.M., Tesfaye, A., Lommen, W.J.M., Lansink, A.O., Tsegaye, A. & Struik, P.C. 2010. Analysis of seed potato systems in Ethiopia. American Journal of Potato Research 87: 537-552. DOI: 10.1007/s12230-010-9164-1
Hunt, D.J. & Handoo, Z.A. 2009. Taxonomy, identification and principal species. In: Root-Knot Nematodes (R.N. Perry, M. Moens & J.L. Starr Eds). pp. 55-97. Wallingford, UK, CAB International.
Hussain, M.A., Mukhtar, T., Kayani, M.Z., Aslam, M.N. & Haque, M.I. 2012. A survey of okra (Abelmoschus esculentus) in the Punjab province of Pakistan for the determination of prevalence, incidence and severity of root-knot disease caused by Meloidogyne spp. Pakistan Journal of Botany 44: 201-207.
Hussey, R.S. & Barker, K.R. 1973. A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Disease 57: 10251028.
Karssen, G., Wesemael, W. & Moens, M. 2013. Root-knot nematodes. In: Plant Nematology (R.N. Perry & M. Moens Eds). pp. 73-108. Wallingford, UK, CAB International.
Kassa, B. 1996. Status of bacterial wilt research in Ethiopia. In: Proceedings of the AHI-Ethiopia Research Planning Workshop. pp. 23-24. Nazret, Ethiopia, AHI.
Kassie, Y.G. 2019. Status of root-knot nematode (Meloidogyne species) and Fusarium wilt (Fussarium oxysporum) disease complex on tomato (Solanum lycopersicum L.) in the central rift valley, Ethiopia. Agricultural Sciences 10: 1090-1103.
Kayani, Z.M., Mukhtar, T., Hussain, A.m. & Ul-Haque, I.M. 2013. Infestation assessment of root-knot nematodes (Meloidogyne spp.) associated with cucumber in the Pothowar region of Pakistan. Crop Protection 47: 49-54.
Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44: 693-695.
Kinyua, Z.M., Olanya, M., Smith, J.J., El-Bedewy, R., Kihara S.N., Kakuhenzire R.K., Crissman, C. & Lemaga, B. 2005. Bacterial wilt disease and the Ralstonia solanacearum species complex. In: Seed-Plot Technique: Empowerment of Farmers in Production of Bacterial Wilt-Free Seed in Kenya and Uganda (C. Allen, P. Prior & A.C. Hayward Eds). pp. 167-175. Saint Paul (MN), USA, APS Press.
Lawrence, G.W., McLean, K.S. & Hankins, G. 1997. Root-knot and reniform nematodes associated with cotton production in Mississippi. In: Proceedings of Beltwide Cotton Conference, Volume 1. pp. 98-99. Memphis (TN), USA, National Cotton Council. Lima, F.S.O., Mattos, V.S., Silva, E.S., Carvalho, M.A.S., Teixeira, R.A., Silva, J.C. & Correa, V.R. 2018. Nematodes affecting potato and sustainable practices for their management. In: Potato - from Incas to All over the World (M. Yildiz Ed.). pp. 107124. London, UK, IntechOpen Ltd. DOI: 10.5772/intechopen.73056 Madden, L.V. & Hughes, G. 1999. Sampling for plant
disease incidence. Phytopathology 89: 1088-1103. Mandefro, W. & Mekete, T. 2002. Root-knot nematodes on vegetable crops in central and western Ethiopia. Pest Management Journal of Ethiopia 6: 37-44. Martin, C. & Nydegger, U. 1982. Susceptibility of tamarillo (Cyphomandra betacea) to Pseudomonas solanacearum. Plant Disease 66: 1025-1027. Mengistie, B.T. 2016. Policy-practice nexus: pesticide registration, distribution and use in Ethiopia. Journal of Environmental Toxicology 2: 1006. Meressa, B.H., Heuer, h., Dehne, H.W. & Hallmann, J. 2014. First report of the root-knot nematode Meloidogyne hapla parasitizing roses in Ethiopia. Plant Disease 98: 1286-1286. DOI: 10.1094/PDIS-04-14-0383-PDN Miheret, S., Seid, A. & Hailu, N. 2019. Distribution and management of root-knot nematodes (Meloidogyne spp.) in tomato (Lycopersicum esculentum) in North Shoa Zone, Ethiopia. Pakistan Journal of Nematology 37: 123-134. DOI: 10.18681/pjn.v37.i02.p123-134 Moens, M., Perry, R.N. & Starr, J.L. 2009. Meloidogyne species - a diverse group of novel and important plant parasites. In: Root-Knot Nematodes (R.N. Perry, M. Moens & J.L. Starr Eds). pp. 1-13. Wallingford, UK, CAB International. Mulatu, E., Osman, E.I. & Etenesh, B. 2005. Improving potato seed tuber quality and producers' livelihoods in Hararghe, Eastern Ethiopia. Journal of New Seeds 7: 31-56. Norton, D.C. 1978. Ecology of Plant Parasitic
Nematodes. USA, John Wiley & Sons Inc. 268 pp. Pagan, C., Coyne, D., Carneiro, R., Kariuki, G., Luambana, N., Affokpon, A. & Williamson, v.M. 2015. Mitochondrial haplotype-based identification of ethanol-preserved root-knot nematodes from Africa. Phytopathology 105: 350-357. Pavithra, S., Ravichandra, N.G. & Nagamma, G. 2014. Interaction of Meloidogyne incognita and Ralstonia solanacearum on on brinjal (Solanum melongena L.). Journal of Plant Protection 7: 411-414.
Reddy, P.P., Singh, D.B. & Kishun, R. 1979. Effect of root-knot nematode on the susceptibility of Pusa purple cluster brinjal to bacterial wilt. Current Science 48: 915-916.
Ryu, E. 1940. A simple method of differentiating between Gram-positive and Gram-negative organisms without staining. Kitasato Archives of Experimental Medicine 17: 58-63.
Sasser, J.N. & Carter, C.C. 1985. Overview of international Meloidogyne project 1975-1984. In: An Advanced Treatise on Meloidogyne. Volume 1: Biology and Control (J.N. Sasser & C.C. Carter Eds). pp. 19-25. Raleigh (NC), USA, North Carolina State University / the U.S Agency for International Development.
sas Institute. 2009. SAS/STAT 9.3 User's Guide: Survival Analysis (Book Excerpt). USA, S AS Publishing. 564 pp.
Schaad, N.W. 1980. Laboratory Guide for Identification of the Plant Pathogenic Bacteria. USA, University of Georgia. 28 pp.
Seid, A., Fininsa, C., Mekete, T., Decraemer, W. & Wesemael, W.M. 2015. Tomato (Solanum lycopersicum) and root-knot nematodes (Meloidogyne spp.) - a century-old battle. Nematology 17: 9951009. DOI: 10.1163/15685411-00002935
Seid, A., Fininsa, C., Mekete, T., Decraemer, W., Wesemael, W.M.L. & Janssen, T. 2019. Biodiversity of root-knot nematodes (Meloidogyne spp.) from major tomato growing areas of Ethiopia. European Journal of Plant Pathology 154: 513-528. DOI: 10.1007/s10658-019-01674-6
Shew, H.D. & Lucas, G.B. 1991. Compendium of Tobacco Diseases. USA, APS Press. 33 pp.
Stewart, R.B. 1956. Some plant diseases occurring in Kefa Province, Ethiopia. pp. 58-60. Alemaya, Ethiopia, College of Agriculture.
Suatmadji, R.W. 1986. Complex diseases involving nematodes and Pseudomonas solanacearum in potatoes in the tropics and subtropics. in: Proceedings
of the International Workshop "Bacterial Wilt Disease in Asia and the South Pacific" (G.L. Persley Ed.). pp. 12G-125. Los Baños, Phillippines, PCARD.
Sundaresh, V.T., Devappa, C.A. & Priya, N. 2G17. Interaction of Ralstonia solanacearum and Meloidogyne incognita on tomato (Solanum lycopersicon L.). International Journal of Current Microbiology and Applied Sciences 6: 156-16G.
Suslow, T.V. & Schroth, M.N. 1982. Rhizobacteria of sugar beets: Effects of seed application and root colonization on yield. Phytopathology 72: 199-2G6.
Taylor, A.L. & Sasser, J.N. 1978. Biology, Identification and Control of Root-Knot Nematodes (Meloidogyne spp.). USA, North Carolina State University / the U.S Agency for International Development. 111 pp.
Tefera, T. & Hulluka, M. 2GGG. Distribution of Meloidogyne incognita (root-knot nematode) in some vegetable fields in eastern Ethiopia. Pest Management Journal of Ethiopia 4: 77-84.
Tessema, L., Seid, E., Woldegiorgis, G. & Sharma, K. 2G2G. Current status of bacterial wilt (Ralstonia solanacearum) disease in major seed potato (Solanum tuberosum L.) growing areas of Ethiopia. Journal of Plant Patholology and Microbiology 11: 497. DOI: 1G.35248/2157-7471.2G.11.497
Waals, J.E., Van Der Krüger, K., Franke, A.C., Haverkort, A.J. & Steyn, J.M. 2G13. Climate change and potato production in contrasting South African agro-ecosystems effects on relative development rates of selected pathogens and pests. Potato Research 56: 67-84.
Wallace, H.R. 1969. The influence of nematode numbers and of soil particle size, nutrients and temperature on the reproduction of Meloidogyne javanica. Nematologica 15: 55-64.
Zia, A., Zia, K., Anwar, S.A. & Iqbal, M. 2G14. Distribution and association of root-knot nematodes (Meloidogyne spp.) with tomato crop in Faisalabad district. Pakistan Journal of Nematology 32: 1-6.
T. Getu, W. Mohammed, A. Seid, T. Mekete, B. Kassa and M. Bogale. Распространенность, встречаемость и характеристика комплекса болезней с участием Ralstonia solanacearum и Meloidogyne spp. на картофеле (Solanum tuberosum L.) в восточной Эфиопии.
Резюме. Было проведено исследование для оценки распространенности, заболеваемости и встречаемости комплекса болезней Meloidogyne spp. и Ralstonia solanacearum (RS) на картофеле. Всего с 92 полей было собрано 92 клубня и 92 образца почвы. Два вида галловых нематод, Meloidogyne incognita (MI) и M. javanica (MJ), и бактериальный изолят RS были идентифицированы отдельно или в смешанных популяциях. Самая высокая распространенность Meloidogyne spp. и комплекса болезней с участием RS составили 87% в Керсе. Наибольшая заболеваемость комплексом болезней составила 70% в Керсе и Чиро. В образцах клубней встречаемость комплекса заболеваний MI, MJ и RS достигла наивысшего среднего значения 84,6% в Керсе. Осведомленность фермеров об этих патогенах кажется хорошей. Исследование подтвердило одновременное появление Meloidogyne spp. и RS у одного хозяина, что указывает на необходимость изучения важности этих патогенов.
