Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops across the world next to potato, McGovern  . The fruits of tomato are popular throughout the world and are used in all kinds of vegetables as raw salad and processed products such as paste and juice. Ripe tomato fruit has high nutritive value and a good source of vitamin A, B, C and minerals, MoARD  . Recently, it started to gain more medicinal value because of its high content of antioxidant including carotenoids, ascorbic acid, phenolics and lycopene, Oduor  . It also serves for export, making a significant contribution to the national economy of Ethiopia with most of the exports to Djibouti, Somalia, South Sudan, Middle East and European markets, Tabor & Yesuf  .
Tomato production reached more than 163.4 million tons cultivated on more than 4.6 million hectares of land worldwide, FAO  . In Ethiopia, there is 6,298.63 hectares area coverage with an annual production of 28,364.83 tones, CSA  . There is also large-scale production of tomato in the central rift valley (CRV) under irrigated and rain-fed conditions having unlimited potentials for expansion if certain production constraints are avoided. Despite its rapid spread across the different localities and agro-ecologies, the production and productivity remain low. Several biotic and abiotic factors are impacting its production. Among the biotic factors, plant diseases caused by plant-parasitic nematodes (PPN) are a costly burden. There are over 4100 species of PPN currently identified, collectively, causing an estimated loss of $80 - $118 billion per year in damage to crops. Off these species, 15% of them are the most economical species directly targeting plant roots of major agricultural crops and prevent water and nutrient uptake resulting in reduced agronomic performance, overall quality and yields, Bernard et al.  . In nature, plants are rarely, if ever, subject to the influence of only one potential pathogen and this is especially true of soil-borne pathogens like fusarium wilt (Fusarium oxysporum) whereby further opportunities exist for interactions with other microorganisms occupying the same ecological niche, Back et al.  .
The combined effect of wilt causing fungi and PPN causes serious damage to different economically important crops worldwide, Mai & Abawi  and Chen et al.  . Based on its worldwide distribution, extensive host range and involvement with fungi, bacteria and viruses in disease complex, root-knot nematodes (RKN) rank first among the top 10 damaging genera of PPN affecting the world’s food supply, Jones et al.  . A Large number of Fusarium wilt fungus affected plants are known to be infected with root-knot nematodes, Goswami and Agarwal  . Fusarium oxysporum, among the Fusarium spp., is the most frequently isolated plant pathogenic fungus on many host plants. Thus, obtaining optimum crop quality and economic production of tomato depends on the development and exploitation of an eco-friendly, sustainable, economical and alternative method of nematode-wilt disease complex management. Being aware of the array of organisms influencing the crop is, therefore, essential, Webster  . Therefore, the main objectives of this research work was to assess the co-existence of Meloidogyne species and Fusarium oxysporum f.sp. lycopersici (FOL) on tomato in the central rift valley of Ethiopia.
2. Materials and Methods
2.1. Description of the Study Area
The study sites, their geographic locations and characteristic features of surveyed tomato fields are summarized in Figure 1 and Table 1. The survey covered different localities belonging to the five districts (Ada-Chukala, Adama, Dugda, Lomie, and Merti) and two commercial farms [African Juice Tibila Share Company (AJTSC) and Nuraera] of Central Rift Valley (located 8˚07' - 8˚43' N, 38˚46' - 39˚59' E) of Ethiopia.
Figure 1. Map showing the sampling districts of tomato-growing areas in the Central Rift Valley, Ethiopia, during 2017/18 cropping season.
Table 1. Characteristic features of surveyed tomato fields in the Central Rift Valley, Ethiopia.
aOnly dominant cropping systems are indicated. A.Chu.—Ada Chukallaa; AJTSC—African Juice Tibila Share Company.
2.2. Sampling and Sampling Units of the Disease Complex
The study areas were purposely selected in consultation with the nearby agricultural offices and development agents (DAs), while the sampling fields were selected randomly. Ten to fifteen mineral (soil) and organic (root & stem base) samples per farm were taken from symptomatic plants to be used as composite sample. Mineral samples were collected from 2 cm diameter next to the plant and top 20 - 25 cm depth to obtain a representative sample making sure not to take the top five centimeters layer, Sasser and Carter  . Areas infested with weeds or other plant species were avoided to prevent sampling nematodes from non-target hosts, Knight  . Mineral fractions were bulked to 2 kg soil per sample. A sub-sample of 1 kg was taken from the composite and packed in a 2 kg size labeled plastic bag. Sub-samples of sufficient amount organic fractions were also packed in separate labeled plastic bags. The hand shovel and other sampling equipment were cleaned and dried after sampling to avoid contamination of soil samples and to prevent the spread of nematodes or pathogens between farms. A total of 118 composite field samples (59 from each fraction) were collected and transported to the plant pathology laboratory of Melkassa agricultural research center. The samples were kept under refrigerator (at 4˚C) until further processing.
2.3. Extraction and/or Isolation and Identification of Pathogens
2.3.1. Extraction and Identification of the Root-Knot Nematode(s)
Nematodes were extracted by Modified Baermann’s funnel, Hooper et al.  technique and identified based on the perineal pattern described by Chitwood  . Infected roots were washed under running water to make it free from soil (Figure 2(a)) and females were dissected out from well-developed galls (Figure 2(b)) of the roots under stereo binocular microscope and transferred to Petri dish containing tap water. The posterior portion of the female was cut with a perineal pattern knife, Taylor and sasser  and the body contents were cleaned.
Figure 2. Infected tomato roots after washing (a) and matured female body from well-developed gall (b) during dissection.
Cleaned posterior portion of female was further trimmed and transferred to a drop of glycerin on a clean glass microscope slide. A cover slip was placed and sealed with nail polish and observed under Leica compound microscope. Ten perineal pattern per sample was processed to determine the frequency of each nematode species. Important morphological characters such as body shape of larvae, males and females were also used during identification. Presence and absence of stylet were used to distinguish pathogenic nematodes from free-living ones.
2.3.2. Isolation and Identification of Fusarium oxysporum
Fusarium wilt disease-inciting pathogen (Fusarium oxysporum) was isolated from organic fractions collected from the survey. Specimens of organic fractions from diseased plant were subjected to running tap water and then rinsed with distilled water. Diseased specimens were cut into pieces (2 cm) and surface-sterilized with 2% NaOCl for 2 minutes, followed by three changes of sterile distilled water and dried in between two sterilized blotting papers. Sterilized and dried specimens were plated out on potato dextrose agar (PDA) medium in sterile Petri dishes. The inoculated plates were incubated at 25˚C ± 2˚C for 7 - 9 days. Identification of F. oxysporum cultures was made on the basis of cultural and morphological characteristics, Leslie and Summerell  followed by pathogenicity test.
2.3.3. Pathogenicity of Fusarium oxysporum
To determine the formae speciales, virulence analysis of the isolates was carried out on a susceptible tomato (cv. Marmande), which has no resistance gene for both Race 0, formerly race 1 and Race 1, formerly race 2, Ibrahim  . Inoculum density (conidia concentration) of the pathogen from 10 days old cultures were adjusted to 3 × 106 Conidia/ml/plant, Lobna et al.  using a hemocytometer. Twenty one days old seedlings were inoculated by standard root dip method, Srivastava et al.  . Seedlings were removed from the pot tray, shaken to remove the adhering particles and washed carefully under tap water. The roots were trimmed with a sterile scissor and were submerged in the conidial suspension for 30 minutes. The inoculated seedlings were transplanted to 20 cm diameter surface sterilized (5% NaOCl) pots, filled with soil, sand and compost in 1:2:1 ratio, and incubated in greenhouse. Four treatments: (1) Marmande + White FOL Isolate, (2) Marmande + Pink FOL Isolate, (3) Marmande + Violate FOL Isolate; and (4) un-inoculated check (inoculated with sterilized distilled water) with five replications was set in the greenhouse. The prevalence and frequency of occurrence of species and/or disease complex were determined using the following formulae based on species identification of the respective pathogens after extraction and/or isolation of the respective pathogens.
2.4. Data Analysis
Data obtained from the survey and greenhouse were subjected to analysis of variance (ANOVA) by GenStat software (16th edition) and statistical package for social sciences (SPSS), respectively and means were separated using LSD at P = 0.05.
3. Results and Discussion
The results of the survey and subsequent laboratory (pathogen identification) and greenhouse (pathogenicity test) works are discussed hereunder.
3.1. Identification of the Test Pathogens
3.1.1. Root-Knot Nematodes (Meloidogyne Species)
Important diagnostic character used for species identification was the perineal pattern found in the female Meloidogyne species. The perineal pattern cut showed high squarish, round to flat dorsal arch. The lateral ridges were absent in many of the samples and few in others. The striae were coarse, smooth to wavy marked with breaks and forks (Figure 3). These characters were compared with the descriptions given by Eisenback et al.  and were inline. The female body was pear-shaped with no visible protruding posterior. The posterior was globose and
Figure 3. Female perineal pattern of RKN during identification; (a) M. incognita and (b) M. javanica.
the neck projects anteriorly (Figure 4(d)). Characteristic offset head shape and narrow and conical, tapering to a finely rounded hyaline tail tip (Figure 4(a) & Figure 4(b)) was also noted during the identification. The stylet was evident (Figure 4(c)) and it affirms, as the nematode was not free-living. M. incognita and M. javanica were the predominant species found in the survey areas (on the basis of perineal pattern characterstics). This result is also in line with previous reports of Seid et al.  and Seid et al.  .
3.1.2. Fusarium Wilt (Fusarium oxysporum f.sp. lycopersici)
Diseased plant organic specimens from survey were used to isolate (identify) wilt-causing pathogen, it was found mainly Fusarium oxysporum and affirm with pathogenecity test for its formae speciales. Pure cultures of 7-9-days-old were morphologically characterized. Morphological characteristics described by Leslie and Summerell  and Rafai et al.  , such as colony color, colony growth pattern, size of macro- and micro-conidia and absence and presence and number of septation of both macro-and micro-conidia were mainly used to narrow down the identification under laboratory condition. The mycelium was found to be floccose (abundant) with three morphotypes or colors (white, pink and violet) as indicated in Figure 5, which was used to separate isolates in the pathogenicity test.
The length*breadth of macroconidia usually varied between (15.9 - 46.98 × 1.83 - 4.88) µm and that of microconidia was (6.75 - 13.56 × 1.93 - 3.4) µm under 40× magnification. The number of septation of microconidia ranges from 1.5 - 4.3 µm. The shape of macroconidia varies from straight, slightly curved to sickle-shaped. The shape of micro-conidia was oval, elliptical or kidney and usually 0-septated with infrequent occurrence of a single septation. False head structures with short monophialides were there in the aerial mycelium, while it was observed under compound microscope without disturbance of the existing mycelium (Figure 6). This result is in line with the description of Fusarium species of Rani and Ramya  & Leslie & Summerell  .
Figure 4. Light micrograph (40×) of the tail (a), head (b), stylet (c) and female Meloidogyne species (d).
Figure 5. Morphotypic isolates of 5-days-old pure culture Fusarium oxysporum; White (a), pink (b) and Violet (c) PDA media.
Figure 6. Macro- and micro-conidia (a), false heads (b) and conidiogenous cells (c) of 10-days-old Fusarium oxysporum on PDA media under 40× magnification.
3.1.3. Pathogenicity of Fusarium oxysporum
Various symptoms on aerial parts and within the stem tissues of tomato plants infected with F. oxysporum were noted starting at 33 days after innoculation (DAI). Yellowing of the lower leaves at early stage of the plant and leaf necrosis and later, dropping due to the infection were the most prominent symptoms. However, there was no statistically significant difference in virulence among the isolates in mean disease severity and area under disease progress (Table 2). As expected, however, there was significant difference (p < 0.05) between the
Table 2. Virulence analysis of the FOL isolates measured by mean disease severity and AUDPC.
Where, means followed by the same letter (s) within the column in each parameter are not significantly different at 5% level of significance; LSD: Least significant difference; CV: coefficient of variation and AUDPC: area under disease progress curve.
inoculated treatments and the un-inoculated check regardless of the isolates. Moreover, successful re-isolation of the pathogen from artifially inoculated symptomatic organic tissue affirms as the pathogen was pathogenic to tomato. Even though, there is no statistically significant difference (p < 0.05) in mean disease severity and AUDPC, numerically higher disease severity and AUDPC were noted from whitish isolate.
3.2. Distribution and Prevalence of Meloidogyne Species and Fusarium oxysporum f.sp. lycopersici Disease Complex
The percent prevalence of the disease complex in this study was determined based on species identification of RKN and fusarium wilt after extraction and/or isolation of the respective pathogens under the laboratory condition. The results of the survey showed the co-occurrence of Meloidogyne species and F. oxysporum f.sp. lycopersici in all assessed areas in the CRV of Ethiopia. This study provides the quantification of occurrence of Meloidogyne species and FOL disease co-occurrence on tomato crop.
Root-knot nematode species morphologically identified from infected roots and rhizosphere soil included M. incognita and M. javanica, which occurred either separately or concurrently. The result showed that 39% of tomato fields were infected with the disease complex (MI*MJ*FOL); off which, 16.9% and 6.8% resulted from concomitant infection of MI*FOL and MJ*FOL, respectively (Figure 7). The prevalence of the disease complex (MI*MJ*FOL) varied in surveyed districts with the highest at Dugda (60%) and the lowest at Lomie, 27.3% (Figure 7). The prevalence of Meloidogyne species and FOL co-occurrence has not been reported before in Ethiopia. However, mixed population of M. ethiopica (32.7%) and M. incognita with M. javanica (3.9%) have been reported, Mandefro and Mekete  and Abebe et al.  . The remarkable co-occurrence of these pathogens in tomato crop grown in the CRV of Ethiopia depicts sever direct or indirect damage to this crop.
The mere co-existence of the two pathogens within a single host plant does not necessarily depict their synergism and therefore, there is a need to investigate
Figure 7. District-wise variation in prevalence (%) of type of species and/or disease complex. Where, ADCH: Adaa chukala and AJTSC: African Juice Tibila Share Company.
their nature of interaction. However, greenhouse experiments elsewhere in the world reported the synergistic effects of different soil-born pathogens and Meloidogyne species on a range of host plants, Al-Hazmi and Al-Nadary  ; Ansari et al.  ; Golden and van Gundy  ; Kumar  ; Kumar  ; Lobna et al.  and Onkendi  . Fusarium wilt-RKN disease complex is one of the most widely recognized and economically important diseases in the world, Wanjohi et al.  ) as they both affect the function of the root system. The two often infect tomato simultaneously, forming a complex that increases the incidence and severity of Fusarium wilt, Onkendi et al.  . Presence of Meloidogyne species greatly reduced resistance of tomato cultivars to the fusarium wilt. Among the Meloidogyne species, M. incognita is the most common in tomato production worldwide, Gautam et al.  . The presence of fungal survival structures (chlamydospores) that enhance its ability to persist in the soil for a long time and its wide host range makes its (F. oxysporum) management fairly challenging, Wanjohi et al.  .
3.3. Frequency of Disease Complex
Disease complex involving MI*MJ*FOL was the most frequent (51%) followed by MI*FOL (22.2%) and the least was MJ*FOL, 8.9% (Figure 8). Out of 59 samples, 51 samples were found carrying Meloidogyne species. There was the highest absolute (16.9%) and relative (22.2%) prominence of MI*FOL as compared to MJ*FOL (6.8%, 8.9%, respectively). This probably indicated that M. incognita appears to be the most important species associated with damage inflicted to tomato production in the area due to the disease complex as it was the frequently found species.
Figure 8. Absolute and relative frequency of occurrence of species and/or disease complex in five districts and two commercial tomato farms of CRV during 2017/18 main cropping season.
Figure 9. Prevalence of Meloidogyne species and Fusarium oxysporum f.sp. lycoppersici in the survey area in 2017/18 main cropping season.
3.4. Status of Meloidogyne Species and Fusarium oxysporum f.sp. lycopersici
The prevalence of Meloidogyne species and Fusarium oxysporum f.sp. lycopersici regardless of their co-occurrence is summarized in Figure 9. There was high prevalence of Meloidogyne species and F. oxysporum f.sp. lycopersici (89.8%, 71.2%, respectively). It is evident from the figure that Meloidogyne species was prevalent (up to 100%) in most the areas assessed. On the other hand, maximum (90.9%) prevalence of FOL was recorded from Lomie district while the minimum (54.5%) was from Adaa Chukala district. This result probably showed high chance of getting synergistic reaction (if it is their nature) or any kind of disease interaction provided that other conducive environmental factor for the complex to occur.
The present survey revealed the existence of the disease complex (co-existence of Meloidogyne species and Fusarium oxysporum f.sp. lycopersici) in the central rift valley of Ethiopia. The mere co-existence of the disease complex in the same host plant with similar niche will not be a sole evidence for the existence of their synergism. However, the pathogens were pathogenic to tomato and indicated the existence of some type of interaction between them. Therefore, the nature of their interaction need to be investigated in the fiture.