The Ethiopian staple food crop enset (Ensete ventricosum) assessed for the first time for resistance against the root-lesion nematode Pratylenchus goodeyi

Selamawit A. KIDANE 1,2, Beira H. MERESSA 3, Solveig HAUKELAND 4,5, Anne Kathrine (Trine) HVOSLEF-EIDE 1,∗ and Danny L. COYNE 2,6 1 Norwegian University of Life Sciences, NMBU, P.O. Box 5003, 1432 Ås, Norway 2 International Institute of Tropical Agriculture, IITA, PMB 5320, Ibadan, Nigeria 3 Jimma University College of Agriculture and Veterinary Medicine, P.O. Box, Jimma, Ethiopia 4 The Norwegian Institute of Bioeconomy Research, NIBIO, P.O. Box 115, 1431 Ås, Norway 5 International Centre of Insect Physiology and Ecology, P.O. Box 30772-00100 Nairobi, Kenya 6 Nematology Research Unit, Department of Biology, Ghent University, Campus Ledeganck, Ledeganckstraat 35, B-9000 Ghent, Belgium

Ensete ventricosum is a large herbaceous plant that belongs to the Musaceae family, the same as bananas. The genus Ensete comprises seven species (E. ventricosum, E. homblei, E. livingstonianum, E. superbum, E. perrieri, E. lecongkietii and E. glaucum) (Cheesman, 1947;Simmonds, 1962;Luu et al., 2012). Wild E. ventricosum species are found distributed in sub-Saharan Africa and Asia, but it is domesticated and cultivated as a food crop only in Ethiopia. Unlike banana, enset does not produce edible fruit, but rather the pseudostem and corm are harvested after 3-12 years and processed into food products. Major food products prepared from enset are kocho (obtained through fermentation of decorticated leaf sheath and corms), bulla (powder from the liquid squeezed out of leaf sheath and pulverised corm) and amicho (boiled corms) (Brandt et al., 1997). In the south and southwestern part of the country, enset serves as a key staple food crop for about 20% of the Ethiopian population (Borrell et al., 2019). It is also important as the key signature crop of the complex enset-based cropping systems in this area, creating stability in relation to food security, as well as the agroecology. As a perennial crop that can be harvested at any time of the year, enset offers food security when other crops are less available, providing a year-round availabil-ity of nutritious food. It is also generally perceived to tolerate drought, with a broad agroecological distribution and is easily cultivated around the home with low input and management requirements. Consequently, the crop represents an important position in household food security. In Ethiopia, enset is reported to be more productive per unit area than other starch crops (Tsegaye & Struik, 2001). In addition to food, enset is also used for a multitude of other purposes, such as for feed, medicine, building and fibre. As an orphan crop, with restricted geography, it has received relatively limited attention in terms of crop improvement. This is beginning to change, however, as the importance of this crop becomes better understood, with a few genetic diversity studies being undertaken, as well as research to identify pest and disease resistance (Brandt et al., 1997;Harrison et al., 2014;Borrell et al., 2020).
More than 600 enset landraces collected from major enset-growing areas in Ethiopia have been conserved ex situ in the gene bank in the Areka Agricultural Research Center (Yemataw et al., 2017). Molecular characterisation of enset landraces using amplified fragment length polymorphism (AFLP) (Negash et al., 2002), random amplified polymorphic DNA (RAPD) (Birmeta et al., 2002, simple sequence repeat markers (SSR) (Olango et al., 2015;Gerura et al., 2019) and inter-simple sequence repeat (ISSR) (Tobiaw & Bekele, 2011) techniques have revealed high genetic diversity amongst various landraces. Despite the progress in genetic studies and the potential of the crop, genetic improvement and conservation are based on conventional methods and have remained stagnant (Olango et al., 2015). To date, breeding enset using conventional or biotechnology applications has yet to materialise in improved varieties for any trait (Merga et al., 2019). Its perennial life cycle, with its extended duration to flowering and seed set, its complex vernacular naming, the absence of known traits such as disease resistance and reliance on vegetative propagation make genetic improvement tedious, expensive and time consuming (Olango et al., 2015). Consequently, enset is by far the least studied food security crop (Borrell et al., 2019).
Despite its resilience and versatility, several production constraints, including plant-parasitic nematodes, challenge enset. Studies have shown that although a range of nematode species are associated with the crop, the rootlesion nematode Pratylenchus goodeyi, root-knot nematodes (Meloidogyne spp.) and the foliar nematode Aphelenchoides ensete appear the most important nematode threats (Peregrine & Bridge, 1992;Swart et al., 2000;Bogale et al., 2004;Addis et al., 2006). However, P. goodeyi is by far the most common and prevalent species, occurring in all fields sampled, at densities as high as 25 000 (10 g soil) −1 (Bogale et al., 2004;Addis et al., 2006;Kidane et al., 2020). When challenged with densities this high, the crop might undergo considerable stress, with roots straining to maintain water and nutrient supply to the plant. However, the damage potential to enset by these nematodes is yet to be determined, as is the susceptibility to nematodes of the various land races and cultivars used by farmers.
Of the various strategies for the management of nematodes, the use of resistance is most suited for smallholder farmers in Africa, but knowledge of nematode pests and their management is poor and access to, or availability of, quality inputs is limited (Coyne et al., 2009). Commercial banana plantations have mainly relied on chemical nematicides, which are not an option for smallholder enset farmers. Exploiting resistance is an alternative management strategy against nematodes (Speijer & De Waele, 1997). Traditional breeding for genetic traits in members of the Musaceae, however, is fraught with numerous obstacles based on inherent sterility, low genetic base and the long-term nature of the crop (Ortiz, 2011). A first step for the development of a management option is to identify cultivars that are resistant to pests and diseases (Speijer & de Waele, 1997;Pinochet et al., 1998;Coyne & Tenkouano, 2005). To date, there has been no known screening for resistance of enset against plant-parasitic nematodes. Resistance is defined as the ability of a host plant to suppress nematode reproduction and development. Whereas nematodes will reproduce on a susceptible host and cause damage, a tolerant host will support nematode reproduction but suffer limited injury even in the presence of high infection levels, while a sensitive host cannot support even a light infection of nematodes (Bos & Parlevliet, 1995).
The objective of our study was to screen and evaluate the host plant response of nine enset cultivars to inoculation with P. goodeyi, in order to identify sources of resistance in the enset germplasm for potential use in nematode management, as well as to assess the pathogenicity of P. goodeyi on three selected enset cultivars.

Materials and methods
All experiments were conducted in the screenhouse located at Jimma University College of Agriculture and Veterinary Medicine, Jimma, Ethiopia, 7°42 N, 36°50 E, at an altitude of 1710 m a.s.l. The area receives an annual rainfall of 1250 mm, average maximum and minimum temperatures of 26°C and 11°C, and an average maximum and minimum relative humidity of 91.4 and 37.9%, respectively.

ENSET CULTIVARS
One-year old enset seedlings, of known cultivars, were obtained from Areka Agricultural Research Centre, Areka, Wolaita. Suckers for each cultivar were regenerated from a single corm to ensure the purity of each cultivar. Prior to planting, roots were removed and the corms peeled before sanitising in boiling water treatment for 20 s (Coyne et al., 2010). The suckers were then trimmed in order to ensure uniformity in size prior to planting. The waste root and corm material was assessed for nematodes before and after boiling water treatment.

NEMATODE INOCULUM
Pratylenchus goodeyi was isolated from infected enset roots collected from a high infection 'hotspot' highland area in Agena, Guraghe, identified during a recent study (Kidane et al., 2020). A combination of morphometric and molecular data revealed that P. goodeyi was the only species of the genus identified from this area (Kidane et al., 2020). Due to there being no monoxenic cultures of P. goodeyi available, naturally infected roots were used as inoculum, which has previously been shown to be a successful alternative (Coyne et al., 2010). Monoxenic culturing of some species of Pratylenchus is also not always successful using the conventional method on carrot discs (Santos et al., 2012), and P. goodeyi has proved difficult to date (Coyne, pers. comm.). Nematodes used for inoculation (P i ) were extracted from a 10 g subsample of chopped enset root and corm material using a modified Baermann extraction method over 48 h (Hooper et al., 2005). Nematodes were collected on a 38 μm sieve, rinsed into beakers, the suspension was reduced to 10 ml, and counted from 1 ml aliquots under a compound microscope.

RESISTANCE SCREENING
Nine cultivars were selected and assessed for resistance to P. goodeyi: 'Gewada', 'Zereta', 'Maziya', 'Heila', 'Kellisa', 'Gefetanuwa', 'Yanbule', 'Messana' and 'Endale'. These cultivars are among the 623 enset accessions maintained in Areka Agricultural Research Centre, obtained from single corms of each cultivar. These cultivars have distinct phenotypic variations. They are among the released cultivars for desired characteristics, such as yield and bacterial wilt disease tolerance. The experiments were conducted on raised benches in the screenhouse using 2 l pots containing oven-sterilised sandy soil, arranged in a randomised complete block design (RCBD) with six plants per treatment (cultivar). Suckers were maintained for 2 months to enable enough root development before inoculation with nematodes. At 2 months after planting (MAP) 2000 P. goodeyi (mixed juvenile and adult stages) were added to the pots in a 7 ml suspension into three holes made using a pencil around the base of the suckers and then covered. The plants were watered as needed and fertiliser applied as urea, once at 3 months after inoculation (MAI). The experiment was terminated at nine MAI and repeated once; the repeat was terminated at 4.5 MAI (due to the availability of seedlings at a later time and timeline of the study period).

PATHOGENICITY ASSESSMENT
Three enset cultivars ('Maziya', 'Arkiya' and 'Heila') were used to assess P. goodeyi pathogenicity. These cultivars are among the cultivars released for their desirable traits and they were also selected, based on results from previous nematode surveys, for supporting either high or low P. goodeyi densities. Enset suckers were planted into 2 l pots and inoculated with 500, 1000 and 2000 P. goodeyi in a 10 ml suspension and compared with a non-inoculated water control. The pots were prepared and maintained as outlined above in the screening experiment, arranged in a RCBD with four plants per treatment (cultivar × inoculum density) on raised benches in the screenhouse. The experiment was terminated at 4.5 MAI; unavailability of seedlings prevented a repeat.

GROWTH AND DAMAGE PARAMETERS ASSESSED
At harvest the plant height, shoot fresh weight and root fresh weight were recorded for each plant. Plant height was measured from the soil surface to the tip of the youngest growing leaf. Plants were carefully removed from pots, rinsed free of soil and dabbed dry with paper towels. The roots were removed with a knife and the shoot (including leaves) and roots weighed separately. Roots were chopped into ca 0.5 cm pieces and nematodes extracted from a 10 g sub-sample per plant. The soil from each pot was thoroughly mixed before removing a 100 ml sub-sample. Nematodes from roots and soil were extracted using a modified Baermann method for 48 h (Hooper et al., 2005). Nematodes were collected on a 38 μm sieve, rinsed into beakers, suspensions reduced to 10 ml and densities assessed from 3 × 1 ml aliquots under the microscope. The overall nematode root and soil densities per plant were calculated by multiplying the density per g root by the total root weight and per ml soil by soil volume (2000 ml). Final nematode population density (P f ) per plant was calculated as the sum of the root and soil factions. The reproduction factor (RF) was calculated by dividing P f by the initial nematode population density (P i ).

STATISTICAL TREATMENT OF DATA
All data were analysed using RStudio ® . The least significance difference was calculated for separation of means with P 0.05. Nematode population densities were log(x+1) transformed prior to analysis of variance in order that data conformed to a normal distribution. Mean nematode population density data were back-transformed for presentation.

PATHOGENICITY ASSESSMENT
Results showed that in the pathogenicity study P. goodeyi multiplied on all three cultivars ('Maziya', 'Arkiya' and 'Heila') after 4.5 months but with no differences in P f or RF among them. Significant differences (P < 0.001) on the P f and RF were observed, based on the four levels of inoculation densities used within each cultivar. We also found that the RF of P. goodeyi was low in all three cultivars compared to susceptible cultivars such as 'Gefetanuwa' as seen in the cultivar screening experiment (Table 2). No differences in plant growth parameters were observed between the controls and inoculated plants (Table 3).

Discussion
Our study represents the first proper assessment of nematode resistance in enset. Although data from a small number of survey studies indicate possible differences in susceptibility or resistance to plant-parasitic nematodes among enset cultivars (Bogale et al., 2004), there is as yet no information available from any controlled studies. Indeed, there is only limited information on the resistance of enset cultivars against the various pest and diseases. Our results reveal that there does appear to be quite a range in susceptibility to P. goodeyi among enset cultivars. The low multiplication of P. goodeyi on 'Maziya', 'Heila' and 'Arkiya' also demonstrates a good level of resistance, with a low population build-up, while 'Gefetanuwa' was highly susceptible, with a much greater P. goodeyi multiplication.
There are over 600 enset cultivars maintained in the Areka gene bank, with a number of studies underway to characterise enset germplasm for genetic and phenotypic variability amongst accessions (Yemataw et al., 2017;Gerura et al., 2019). Screening activities, such as the current study, help contribute to building up the information on the various accessions, towards detecting sources of resistance across a range of constraints and identifying suitable sources for breeding. The current study initiates information gathering for nematode resistance and shows some promising results that provide a basis for further large-scale screening studies. However, the process is time consuming and subject to sensitivity and error, while ambiguity of accession names can be misleading. Conse-   Final nematode density analysis was undertaken using log-transformed data; back-transformed data are presented. Mean P. goodeyi densities and RF of each cultivar in a row with the same letter are not significantly different (P 0.05). Mean P. goodeyi densities and RF across three cultivars in a column with the same letter are not significantly different (P 0.05). quently, suitable protocols need to be established, based on the use of accessions that are genetically characterised for conformity of names. Rapid screening procedures targeting single roots and assessing nematode multiplication adopted for banana (De Schutter et al., 2001;Coyne & Tenkouano, 2005) can also be used to screen enset germplasm. The development of tissue culture-based in vitro propagation protocols for enset (Negash et al., 2000) could also improve the efficiency and speed of propagating disease-free planting materials for distribution to farmers.
Determining germplasm with good resistance to key pests, diseases and abiotic constraints is important for improving crop productivity, especially in Africa, where losses are particularly large (Coyne et al., 2018). Identifying accessions that have multiple resistance is therefore of even greater value when determining germplasm for use in breeding programmes, or providing recommendations to farmers. For instance, 'Maziya' is regarded as less susceptible to bacterial wilt disease (Xanthomonas vasicola pv. musacearum), whilst 'Gefetanuwa', which supported the highest reproduction of P. goodeyi, also supports rapid X. vasicola pv. musacearum development, as does 'Arkiya', which has been used as a susceptible control in evaluation studies (Muzemil et al., 2019). Although 'Arkiya' was regarded as one of the cultivars with higher densities of P. goodeyi in a previous survey (Bogale et al., 2004), the P f and RF were similar to the other two cultivars ('Maziya' and 'Heila'). As nematode infection is known to predispose banana to bacterial wilt (Shehabu et al., 2010) and fusarium wilt diseases (Almeida et al., 2018), it further serves a purpose to have nematode resistance traits in banana, as well as enset. Studies such as ours can be very important to identify cultivars to use for studying the relationship of nematodes and bacterial wilt disease.
In our study we found that infection with P. goodeyi did not result in any decrease in growth parameters of the enset suckers over the 4.5 months of assessment, as compared to similar studies with banana ( Van den Bergh et al., 2002;Dochez et al., 2009;Coyne et al., 2013). This could be explained by the long perennial nature of the enset crop, with about 7 years to maturity, and the period of assessment being too short to detect differences. Alternatively, it may be that the enset cultivars assessed in the current study all exhibit a level of tolerance to the nematodes. This may also explain the high P. goodeyi densities experienced on enset roots during recent surveys (Bogale et al., 2004;Addis et al., 2006;Kidane et al., 2020). Similarly, unlike other studies on banana, root necrosis damage was not readily observed or visualised, possibly due to the thin enset roots, combined with the short duration of the experiment, or possibly due to host tolerance. Infection of enset roots with P. goodeyi does result in necrosis, however, which can be considerable, as seen during field studies (Bogale et al., 2004;Addis et al., 2006;Kidane et al., 2020) and which is undoubtedly detrimental to growth and production of enset. In any case, it is clear that further investigations are necessary to determine more effectively host damage potential by P. goodeyi, possibly over a longer duration, and with a greater range of germplasm assessed using methods such as the single-root inoculation (Coyne & Tenkouano, 2005), which should be repeated to confirm results.
Although the current study screened a few cultivars from the enset germplasm and over a short duration compared to the perennial nature of the crop, this study demonstrates that there are indeed differences in the resistance of cultivars to P. goodeyi. Being the first study conducted on enset resistance against nematodes, it can be used as a base for further studies such as screening and interaction of other nematodes and other pathogens.
Most synthetic chemical nematicides have been removed from the market due to environmental and human health concerns and so it is important to select the best performing cultivars in terms of resistance to nematodes and other diseases. Chemical pesticide use on enset is currently very low under the predominantly subsistence manner of production around homesteads. Therefore, the identification of cultivars resistant to the predominant nematode species is a first step towards using those in future breeding efforts.
Despite the importance of enset in Ethiopia, there has been little attention given to the genetic improvement of the crop. Baseline studies on the identification of nematode resistance, such as ours, accompanied by information on the molecular characterisation and genome-wide sequence data of enset (Harrison et al., 2014) will enhance research on this important but neglected crop towards its genetic improvement. Having established tissue culture propagation and in vitro conservation protocols for enset will additionally provide a basis for extending such screening work (Negash et al., 2000;