First Report of Stemphylium vesicarium Causing Onion Stemphylium Leaf Blight in Ethiopia

A suspected fungal disease was observed on onion leaves during survey in onion fields of the central rift valley of Ethiopia in the offseason in 2019. The disease symptoms were round to oval small spots on onion leaves which coalesce to form blighted leaves that change to brown lesion and black sporulation with time. The disease was of high incidence (up to 95.2%) and severity (up to 4.67) where in some fields it has been causing early plant senescence and reduced bulb size during harvest with massive yield loss, named to be “Yeshinkurt Ebola” to mean “Ebola of onion” by farmers. Dirty white to light grey front colony color and light brown to deep brown reverse colony color with alternative white and brown band fungal growth were isolated from infected onion leaf samples. The conidia were olive brown, oval to ovoid, oblong and ovoid to oblong are born on conidiophores. Pathogenicity of the pathogen was confirmed on 45-days-old onion (cv. Nafis) plant, and it was identified as Stemphylium vesicarium based on cultural, morphological and pathogenic results. This is the first report of S. vesicarium, the causative agent of onion stemphylium leaf blight.


Introduction
Many small-and large-scale farmers in the main onion-producing areas of central rift valley of Ethiopia grow onion (Allium cepa L.). In total, 31,673 ha of land were covered by onion with 293,887.6 tonnes of production, CSA [1]. Nowadays, the area under onion production is increasing. However, its production and/or productivity is affected by many biotic (diseases, and insects pests) and abiotic like imbalanced fertilizer, uneven irrigation and inappropriate agro- 1105 Agricultural Sciences nomic practices, Amin et al. [2]. Currently, a strange disease, expected to be stemphylium leaf blight is observed in farmers' field and research stations of the CRV.
In Ethiopia, purple blotch (Alternaria porri) and downy mildew (Peronospora destructor) diseases are considered as the major constraints of onion production and productivity, Wondirad et al. [22]. Purple blotch is prevalent in all oniongrowing areas of the country. It attacks leaves, bulb and seed stalks and subsequently reduces yield and quality. Since 2018, however, a new disease symptom, which confuses with purple blotch disease, was observed in the central rift valley areas of Ethiopia. The disease typically attacks leaf, which starts with round to oval small spots that later coalesce to form blighting to leaf tissue. The lesion turns brown to tan, for some time; the disease was probably misdiagnosed as

Description of the Study Area
The field survey was conducted during the 2019 cropping season. The survey

Sampling and Sampling Units
Fields were ordinarily selected at intervals of 5 -10 km along the main roads.
When necessary, the sample sizes (the number of observed fields per district) and the distance between sample units (the arbitrarily selected quadrants, 5 -10) per field were adjusted to suit crop distribution and field-size. All sampled fields     Virulence test of the isolates recovered was carried out on Nafis onion variety.

Isolation and Pathogenicity Test of the Pathogen
Pure culture of 14-days-old S. vesicarium was flooded with sterile tap water and, after gently rubbing with a rubber spatula, the resulting suspensions were filtered through sterile nylon gauze. Concentrations of the conidial suspensions were determined with a haemocytometer, adjusted to 3 × 10 4 conidia mL −1 , Köhl et al. [7] with sterile tap water to inoculate 45 days old seedling in the greenhouse. The inoculated onion plants were kept up to 45 days in pots in the greenhouse at 27˚C average temprature and 75% average relative humidity.

Symptoms
During the survey, the overall look of onion fields affected by the disease is depicted in Figure 1. Early senescence of leaves and flower stalks was observed, especially, when the severity of the disease was high (Figure 1(a)). Besides, reduced bulb size (Figure 1(b)) and associated yield loss was observed whenever matured onion fields were encountered during the survey. This was further confirmed from the communication made with producers during the assessment.
Due to this massive appearance of the disease symptoms and maximum production loss, we were even told that farmers in the area call it as "Yeshinkurt Ebola", meaning "Ebola of onion". Specific disease symptoms of small to advanced tan to brown, water-soaked lesions on leaves and onion stalks, advancing to dark-brown to black sporulation and whitish fruiting structure were common (Figures 2(a)-(d)). These symptoms were more obvious on older plants. Overall, the symptoms observed during the survey are similar with symptoms description of SLB by Basallote-Ureba et al. [24].

Disease Incidence and Severity
Severity and incidence of SLB in the surveyed area are summarized in Table 1.
Accordingly, the highest (95.2%) and the lowest (10%) mean disease incidence was recorded at Jeju and Adama districts, respectively; while the maximum disease severity (4.67) was observed at Dugda. In general, SLB was prevalent in all districts assessed with various range of incidence and severity. According to the farmers, in areas such as Koka, the SLB disease was started to be observed since 2016, which might have been misdiagnosed as purple blotch disease.

Identification of the Pathogen from Diseased Leaf Samples
Cultural, morphological and pathogenic characteristics of the isolates from symptomatic onion leaf specimen revealed the presence of S. vesicarium.

Cultural and Morphological Characteristics
The in-face colony color was dirty white to light grey (Figure 3(a)). The reverse colony colors were deep brown, brown and light brown with circular white and brown alternate band growth pattern (Figure 3(b)). This dirty white and brown alternate growth and sporulation have found a distinct feature to S. vesicarium colony growth on PDA media. This result is equivalent to pathogen descriptions of Hosna et al. [25]. The conidia were olive-brown, oval to ovoid, oblong and ovoid to oblong, are borne on conidiophores that are pale to brown with dark edges and bands ( Figure 4). This is in line with the S. vesicarium previous description, Simmon [26]; Basallote-Ureba et al. [24] and Woudenberg et al. [15].

Pathogenecity of Stemphylium vesicarium
Patogenicity test was conducted to confirm whether the pathogen is the primary cause of the disease observed on onion fields during the survey. Periodical assessment (i.e. every week after 1 st symptom observation) resulted in small white tan spots starting from 14 days of post-inoculation ( Figure 5). The symptoms  have been intensive and extended to all inoculated plants with in a pot and were as similar to the symptoms observed during the survey. Moreover, successful re-isolation of the pathogen from artifially inoculated symptomatic leaf tissue affirms that the pathogen was pathogenic to onion and that was realy responsible to massive production loss observed during the survey, which the onion farmers in the CRV area call it "Ebola of onion".

Conclusion
Based on the literature, this is the first report of the occurrence of stemphylium leaf blight (S. vesicarium) on onion in Ethiopia. Complementary work to map its distribution and intensity in the country, determining other putative hosts and the best management options will be relevant to manage the disease.