Prevalence, intensity, and morphological variability of wheat blotch (Zymoseptoria tritici) in Oromia, Ethiopia

Article History Received: September 17, 2021 Revised: December 12, 2021 Accepted: December 16, 2021 Studies of the wheat Zymoseptoria tritici blotch (ZTB) status in different locations, on agronomic practice, and pathogen variability has not yet been studied in Ethiopia. As a result, the goal of this study was to determine ZTB's distribution and intensity, as well as the morphological variability of isolates. In Oromia's central-southeastern region, zones and districts were purposefully chosen, whereas kebeles were determined via a systematic sampling procedure. In a generalized linear model (GLM), the mean comparison of fixed effects was examined using least significant difference (LSD) tests. Colony texture, shapes, and colors were used to identify isolate variability. Pearson correlation was used to examine the relationship between disease intensity and the independent variable, and multiple regression analysis was used to estimate the magnitudes of the association. A total of 108 fields were examined, with the percent occurrence of zones (88.9 to 100%) and districts (77.8 to 100%) recorded. ZTB intensity was not significantly different across districts (p < 0.05) while severity was significantly different across zones (p < 0.01). Weed infestation (r = 0.78 and r = 0.20) and growth phases (r = 0.72 and r = 0.36) had a positive correlation, although plowing frequency (r = -0.77 and r = -0.43) had a negative correlation with incidence and severity. There are 43 isolates classified into four colors, three textures, and three growth forms. The ZTB epidemics in current research areas are need more consideration and they should be prioritized for integrated management. Our data suggest that weed control, soil tillage, and crop rotation are all effective ways to mitigate the effects of wheat ZTB.


INTRODUCTION
Wheat Zymoseptoria tritici blotch (ZTB) is a devastating disease that causes problems in many parts of the world (McDonald et al., 2015;Mehra et al., 2018;McDonald and Mundt, 2016;Dalvand et al., 2018). It is a hemibiotrophic fungal pathogen (Zhong et al., 2017) that causes significant yield loss in wheat by disrupting the photosynthetic component of the plant (Griffiths and Ao, 1980;Eyal, 1981). ZTB epidemics in wheat fields are mostly determined by host vulnerability and climatic factors (Eyal, 1987). Inoculum density, strain pathogenicity, and cultural practices are all factors that influence it (Kema and van Silfhout, 1997;HARRAT and BOUZNAD, 2018). The principal inoculums are obtained through diseased plant residue, seeds, and alternate hosts (Ponomarenko et al., 2011;Holloway, 2014;Steinberg, 2015). ZTB infestations have been related to wheat yield losses of 30 to 54% (Eyal, 1987) and even greater than 60% (Shipton et al., 1971). ZTB causes 25 to 82% wheat yield loss in Ethiopia, with incidence and severity increasing in the key production areas (Abebe et al., 2017;Abeyo et al., 2011;Hailu and Woldeab, 2015;Takele et al., 2015;Said and Hussien, 2013). The losses in yield related to severe ZTB occurrences have been found to vary from 31 to 53% (Babadoost and Hebert, 1984) to 56% (Eyal, 1981). ZTB can be found all around the world (Ponomarenko et al., 2011). For the first time, ZTB was discovered in 1956 in Ethiopia (Stewart and Yiroou, 1967). Nowadays, ZTB is distributed in Oromoia, Amhara, SNNPR regions of Ethiopia (Tadesse et al., 2018;Said and Hussien, 2013;Azanaw et al., 2017). Its severity is highest in Ethiopia's central highlands (Ayele et al., 2008;Ababa Tarafa, 2020) and in environments with high humidity, altitude, and warmer temperatures (Azanaw et al., 2017;Eyal, 1987;Ponomarenko et al., 2011;Ghini et al., 2008). The pathogen's diverse population is to account for the high intensity. Z. tritici exhibits distinct growth forms, hues, and textures, according to investigations of colony morphology on various media (HARRAT and BOUZNAD, 2018;Ayad et al., 2014;Bentata et al., 2011). This suggests that the pathogen is very variable among the population due to genetics (Kema and van Silfhout, 1997;Mekonnen et al., 2020). Aside from the assessment, one of the few types of ZTB research done in Ethiopia was the evaluation of fungicides and wheat cultivars under natural infection. However, there have been no morphological or pathogenic variability studies of Z. tritici isolates yet. Because the disease is dynamic, ongoing disease assessment and studies of disease variability are utilized to alert farmers and governments early, devise management practices, and conduct additional research. The goal of this research was to evaluate ZTB distribution and intensity in a previously unstudied location, as well as to identify the variety of collected isolates based on colony colors, growth patterns, and textures.

MATERIALS AND METHODS Description of the survey areas
During the 2019 cropping season, ZTB field surveys were done in central-southeastern Oromia, Ethiopia. Arsi, West Arsi, Bale, and West Shoa zones were all surveyed ( Figure 1).

Sampling method and strategy
From flowering until maturity, wheat ZTB survey was carried out. The four zones and three districts were chosen from the region using a purposive sampling method. At 5-10 km intervals along the main, available, and accessible roadsides, three kebeles within each district and three farms within each kebele were assessed (Table 1). Farmers' training centers and research stations were also surveyed at the same time. Infected wheat leaf tissues were collected, as well as 91 green leaves with pycnidia and a few dried samples from 108 farmers' fields in paper bags for pathogen isolation (Figure 2A and B).

Diseases Assessment
Depending on the size of the field, 1 m 2 quadrant was thrown at three to five spots at random, with 15 meter intervals along the section. Each 1 m 2 quadrant had 14 plants randomly selected and analyzed for ZTB incidence and severity (Eyal, 1987). ZTB prevalence was estimated by dividing the number of infected fields by the total number of fields examined, and incidence was obtained by dividing the number of infected plants by the total number of plants assessed from three quadrants (Cooke, 2006). Severity was measured on a two-digit scale (Saari and Prescott, 1975). The first digit (0-9) represents the ZTB upward migration on the plant, and the second digit (0-9) determines the severity of the total foliar infection on the whole plant (Eyal, 1987). Its severity index was determined by the formula; Where, D1 representing STB upward movement,

A B
whereas D2 is the severity. Y1 represents the maximum ZTB upward movement and Y2 represents the maximum severity (Sharma and Duveiller, 2007). Data of agronomic practice (Table 2), altitude (Table1), and crop growth stage were gathered to do an association with ZTB intensity. The longitude and latitude coordinates of each field were taken using a global positioning system (GPS) ( Table  1).

Isolation process
Isolation was carried out in the Holeta National Biotechnology Research Center's Microbiology Laboratory at Holeta, Ethiopia. With a little modification from the original protocol, the isolation was completed (Eyal, 1987). The filter paper was placed on the Petri plate and wetted with distilled water in the first stage. The wheat leaves were then placed on the wetted filter paper in a 7 cm segment. For enhancing pycnidiospore oozing from an opening of the pycnidium (ostiole), the petridish was incubated at 24 ℃ for 2 to 8 hours depending on the stages of leaves. The produced oozes were transferred to potato dextrose agar (PDA) supplemented with chloramphenicol succinate 250 mg for 1 liter distilled water using a dissecting microscope or stereoscope (Eyal, 1987). Pycnidia that did not generate ooze, on the other hand, were extracted from the leaf epidermis and placed onto PDA plates using a sterile needle. The colony was picked via sterile loops and smeared onto PDA plates after seven days. The streaked plates were incubated for seven days in a 24 ℃ incubation chamber to promote fungal growth. The single pinkish-orange, dark hard color colony that matched (HARRAT and BOUZNAD, 2018) were streaked on PDA plates and then chosen and distributed on new PDA plates without antibiotics.

Colony morphology
On PDA, cultural appearances (colony color, shapes, and texture) were identified based on macroscopic inspection. The colony morphology was described using both a laboratory manual and a graphical atlas for fungal identification (Watanabe, 2010).

Data analysis
The data was analyzed using SAS version 9.3 statistical software (Stokes et al., 2012). The survey data were converted using ARCSINE after Kolmogorov-Smirnov analysis showed the substantial differences (p < 0.05) and exhibited a non-normal distribution (Kema and van Silfhout, 1997). Fixed factors were structured in three phases of nested design (Tsedaley et al., 2016), with the exception of farmers' fields, which were regarded as a random effect (Table 3). Kebeles were nested under districts in the three levels of nested design, while districts were nested under zones. Pearson correlation was used to examine the relationship between ZTB intensity and agronomic practice, altitude, and crop growth phases, and multiple regressions were used to predict the magnitudes of ZTB intensity.

Distribution of Zymoseptoria tritici blotch across a location
Wheat ZTB was found in all of the investigated areas, with prevalence rates of 100%, 88.8%, and 96.3% in Bale, Arsi, and both West Arsi and West Shoare, respectively. The over all of the surveyed zones had the highest ZTB prevalence (95.4%). It was found to be 100% prevalent in eight districts (Tokekutaye, Ambo, Welmera, Adaba, Dodola, Hetosa, Goba, Agarfa, and Sinana) but Lemunabilbilo district having the lowest prevalence (77.8%) (Figure 3).

The intensity of Zymoseptoria tritici blotch across a location
The incidence of ZTB was not substantially different at the zone and district levels (p < 0.05). This indicated that it has infected wheat crops in all of the surveyed areas in a similar manner. West Shoa, West Arsi, Arsi, and Bale zones had ZTB incidences of 95.7%, 94.7.9%, 87.7%, and 99%, respectively. The maximum incidence (100%) was recorded in three districts (Tokekutaye, Dodola and Sinana), while the lowest incidence (75%) was recorded in Lemunabilbilo ( Figure 3). Between the four zones, ZTB severity index revealed highly significant (p < 0.01) differences. The severity indexes of the Arsi and Bale zones were notably different, but the severity indexes of the other zones were similar (Table 4). The severity index of the districts, on the other hand, did not differ substantially (p < 0.05). At district level, Tokekutaye received the highest severity rating of 42%, while Lemunabilbilo received the lowest severity index of 12% ( Figure 3). Association of Zymoseptoria tritici blotch with agronomic practices, altitude, and wheat growth stages ZTB severity score showed a positive correlation (r = 0.78) and a highly significant difference (p < 0.001) with weed infection levels. Plowing frequency was found to have a negative relationship with ZTB severity index (r = -0.77) and incidence (r = -0.43). ZTB severity and wheat crop stages showed strong positive relationships (r = 0.72). According to our current findings, the increase in altitude in meters has no significant relationship with disease severity (p < 0.05) (

Microscopic and Morphological variability
Zymoseptoria tritici isolates produced very thin pycnidiospores with more than three septation and few curves in form. The shapes, size and septa of pycnidiospores of ZTB isolates are the same. The Zymoseptoria tritici isolates were produced macropycnidiospores of very thin, and more than three septation and erect in shape. Also, the isolates were produced micropycnidiospores in those are without septa (Figure 4). Six pinkish colony isolates had a creamy texture and three different growth forms: dense, medium, and sparse. The whitish color isolates had a creamy texture, and the ooze floods the sowing lines. On PDA, darkcolored isolates grow compactly, densely, and sparsely. Brown color isolates have an intermediate, solid, and creamy texture, with sparse and thick growth patterns (Table 7 and Figure 5). Only two (4.5%) of the total isolates showed whitish colony color. A colony of black color was composed of 28 isolates (63%) of the total isolates, and this colony became the most dominant. Out of the total isolates analyzed, 8 (18.2%) have a brown color and 6 (14%) have a pinkish color.   The colors of nine isolates generated from Bale samples varied. Four isolates were pinkish in color, three were brown, and two were black in color. The isolates were taken from the Arsi zone, and one was whitish, three were black, and two were pinkish. One brown and five black colors were found in West Arsi isolates. Eighteen isolates from West Shoa produced black colonies, while one and four isolates produced pinkish and brown colonies, respectively (Table 7 and Figure 5). A total of 44 Z. tritici isolates were obtained from 91 samples collected across the Oromia region (Table 8 and Figure  6).

DISCUSSION
The significant prevalence of ZTB in the examined locations can be attributed to favorable environmental conditions for ZTB development (regular rains and mild temperatures) (Gilchrist and Dubin, 2002;Teklay et al., 2015). In the altitude range of 2072 to 3043 m.a.s.l, (Tadesse et al., 2018) reported a 38 to100% ZTB incidence. Furthermore, the current findings demonstrate that ZTB is found in 100% of the assessed locations, indicating that it is a severe danger to wheat production in the country. The ZTB disease is very important in the entire world. Argentina, Ethiopia, Iran, the United States, the Netherlands, Russia, New Zealand, and Australia are among the largest wheat-producing countries on the planet. In Iran, Tunisia, and Morocco, it is a major issue with durum wheat (Ponomarenko et al., 2011;Eyal, 1987). High inoculum levels associated with farming methods, particularly in the examined areas, are thought to be the cause of the high incidence. Farmers, in general, do not use appropriate crop rotation systems with nonpathogen host plants and cultivate wheat from year to year, particularly in the Arsi and Bale zones. Because it overwinters in the soil and decaying plant residues as pycnidia, has a higher chance of inoculum survival (Ponomarenko et al., 2011). The high ZTB incidence found in this study is due to high inoculum build up, susceptible cultivars planted by farmers, and favorable environmental conditions across all agro-ecologies in the examined areas of the country. Crop rotation with non-host crops was not practiced by the majority of farmers in the examined area, regardless of zone, and poor weed management and low plowing frequency were also prevalent. In comparison to Ethiopia's central highlands, monocropping is typical in the Arsi and Bale zones. Greater weed population can exacerbate the severity of ZTB. This could be due to wheat competing with weeds for nutrients, water, space, and sunlight, resulting in increased wheat succulence and less ability to resist the pathogen physically (Agrios, 2012). The plant's canopy draws the wheat leaves closer together, making it simpler for rain splashes to disperse spores and altering the pathogen's life cycle (Ponomarenko et al., 2011;Eyal, 1987). In dense plant population, the microclimate, such as high moisture, was always present, providing a favorable setting for the disease. It is possible that the higher plant density leads to a more suitable microclimate within the leaf canopy, which promotes ZTB development (Ansar et al., 2010). Many researches on the impact of environmental conditions on Z. tritici have found that temperature fluctuations play the most crucial function. The Z. tritici body temperature is the wheat leaf temperature that develops into plant leaves, influencing their life cycle significantly (Pietravalle et al., 2003;Gladders et al., 2001;Lovell et al., 2004). Aside from temperature, moist leaf surface plays a significant role in early infections, necessitating a total of 10 mm of rain during three consecutive rainy days with at least 1 mm of rain (Pietravalle et al., 2003). The severity of ZTB reduced as the frequency of plowing increased, and similar trend was seen with Z. tritici (Bailey et al., 2001;Fernandez et al., 2016;Bankina et al., 2014). The effects of soil tillage on ZTB have been researched in a variety of locations. The severity of ZTB was higher in plowed plots under conventional tillage (Gilbert and Woods, 2001;Bürger et al., 2012;Fernandez et al., 2016) than in alternative tillage systems, notwithstanding the contradicting results. Increased soil tillage is utilized for a variety of reasons during crop cultivation, including exposing inoculum to sunlight and removing inoculum sources from the soil. As a result, reducing the amount of inoculums in the soil may hinder the ZTB life cycle (Fernandez et al., 2016;Mergoum et al., 2007). As the frequency of plowing increased, the incidence of ZTB reduced once more. Rotation to non-hosts and agricultural debris sanitation achieved by deep plowing can reduce the quantity of inoculums available to start a new ZTB life cycle. Due to the long-distance spread of ascospores, this may be less effective in the field, but it may be beneficial if used within a region (Ponomarenko et al., 2011). Some research have found a low incidence of ZTB under zero tillage or conservation tillage, but this outcome varied (Gilbert and Woods, 2001). The incidence of tan spot and powdery mildew is reduced as the plowing frequency is raised in farmer's fields, but the incidence of ZTB is increased (Krupinsky et al., 2007). Conservation tillage is encouraging the over-summering of Z. tritici, according to (Mergoum et al., 2007). Throughout the survey effort, the majority of the district's wheat growth stages were at the dough stage. Although the crop had reached full maturity in certain districts, particularly in the midlands. The severity of ZTB was influenced by the variation in growth stages. Because of senescence, the positive association shows that as the crop stage progressed, the severity of the ZTB increased as well. The reason for this is because as the crop matures, it loses its physical and chemical defenses, allowing the disease to easily penetrate and develop on the crop (Agrios, 2012). The different reports showed that the increment of altitude in meter negatively correlated with wheat stem rust (Hirpa, 2018) but from our study, the ZTB intensity is not correlated with altitude in the surveyed areas. We measured disease severity and concluded that when plowing frequency increased, disease incidence decreased by 11.84%. As weed infection levels increased, disease severity increased by 9.73%. Conversely, as plowing frequency increased, disease severity decreased by 10.42%. Other effects included an increase in disease severity of 3.19% as crop growth stages progressed from flowering to maturity. Zymoseptoria tritici pycnidiospore differed from Parastgnospora nodurum pycnidiospore, which were thick, had less than three septations, and had an erect morphology. The germinated spores of the Septoria tritici isolates had the different number of septations, shape, and thickness from Parastgnospora nodurum isolates (Eyal, 1987). On a solid PDA media, the colony morphology of 44 isolates revealed a wide range of textures, growth patterns, and colors ( Figure 5). The whitish color isolates were discovered in the current experiments and had never been reported before (HARRAT and BOUZNAD, 2018). EtAm-14 and EtA-4 had the pinkish color similar to Bale Zone and EtA-3, EtA-8, and EtSh-1 also had the black color similar to the West Shoa zone. This indicates that location may not affect the outcome of colonies of various colors resulting from isolates plated on PDA media, meaning that isolates collected from different locations and plated on PDA media could have the same or various colors, or isolates from the same location had different colors and from the same causative agent (Saidi et al., 2012). When Z. tritici isolates were plated on PDA growth media, they showed morphological differences.

CONCLUSION
The Z. tritici disease was prevalent in the most of the wheat production areas and it intensity also very high in most of the areas where wheat production is known such as Bale, Arsi and west arsi Ethiopia. Furthermore Z. tritici has a wide range of colony shape, which is new to our country. The morphologic heterogeneity of wheat Z. tritici isolates in Ethiopia was validated by the current finding. Because wheat Z. tritici is extremely common and severe in all of Ethiopia's central-southeast regions, and wheat is the country's most important crop, focusing on building an effective ZTB management strategy is crucial.

ACKNOWLEDGMENTS
The authors gratefully acknowledge the Ethiopian Institute of Agricultural Research for financial support of the study, Kulumsa Agricultural research center, and all participants during the survey program.

DATA AVAILABILITY
The data that support the findings of this study are available on request from the corresponding author.

ETHICAL STATEMENT
This study did not engage in any human or animal testing.