Fungicide evaluation against head smudge (Curvularia spp.) and its impact on yield and germination of tef (Eragrostis tef Zucc.)

doi:10.21203/rs.3.rs-4874832/v1

Head smudge, caused by Curvularia spp., is the major disease of tef in the humid tef growing areas of Ethiopia. Despite this, farmers continue producing tef without any disease management strategies, which is leading to substantial quantitative and qualitative losses. Hence, the field and laboratory experiments were conducted to assess the efficacy of seven fungicides for the management of head smudge and simultaneously to assess the possible effects of the disease on seed quality of tef. The field experiment was conducted at Dangila and North Achefer districts and the treatments were arranged in a randomized complete block design with three replications. Tef seeds harvested from field experiments were subsequently used for the laboratory studies using the blotter method and arranged in a completely randomized design. The effect of fungicides on reducing disease incidence and severity and increasing yield was significant (P < 0.001). The highest overall mean disease control (88.9%) was obtained from propiconazole sprayed plots followed by trifloxystrobin + tebuconazole and tebuconazole, both of which had a similar 85.5% disease reduction. The applications of triadimefon resulted in intermediate disease reduction (69.9%). However, the application of mancozeb + cymoxanil, cymoxanil + copper oxychloride, and thiamethoxam + metalaxyl + difenoconazole doesn’t bring a significant improvement from the untreated plot. According to our findings, head smudge can reduce tef yield by up to 62% and germination by 18.7 to 45.4%. Therefore, application of propiconazole, trifloxystrobin + tebuconazole and tebuconazole can be considered as effective head smudge management practice until other management options are developed.

Fungicide

head smudge

severity

yield

germination

Tef is a C4, self-pollinated, chasmogamous annual cereal. It has a fibrous root system with mostly erect stems, while some varieties are bending or elbowing types (Ketema, 1993). Tef (Eragrostis tef (Zucc.) Trotter) belongs to the family Poaceae, and genus Eragrostis (Costanza, 1974). The genus Eragrostis is the biggest genus from the grass family, contains about 350 species (Watson and Dallwitz, 1992). Of those 54 species found in Ethiopia, 14 are endemic to the country (Cufodontis, 1974). Thus, it is not debatable that, Ethiopia is the center of both the origin and diversity of tef (Vavilov, 1951).

Tef, is the most important cereal of Ethiopia, accounting for about 23.8% (3 million hectares) of the total grain crop area of 80.7% (10 million hectares) (CSA, 2018). For millennia, farmers cultivate tef mainly for the preparation of the best quality and most consumer preferred traditional dishes known as injera. The straw is a good source of animal feed and serves as a binder of mud used for plastering walls of local houses, and as bedding and mulch material. The crop has a great economic value due to its wide agroecological adaptation, reasonable tolerance to drought and waterlogging, minimal post-harvest losses and high storage longevity (Assefa et al., 2017). Nutritionally, tef is a very nutritious cereal grain, rich in amino acid, lysine, and minerals (Mengesha, 1965; Jansen et al., 1962; USDA, 2015). Tef is free of protein known as gluten which can cause gluten protein allergy ailments known as celiac disease (Spaenij-Dekking et al., 2005).

Despite its various importance, the national productivity of tef (1.66 t/ha) is still very low compared to the other cereals belonging to the same family grown in Ethiopia (CSA, 2018). This is because extensive production of low yielding varieties coupled with unimproved traditional production techniques. Lodging, abiotic and biotic factor’s including weeds, diseases and insect pests, are among the major factors limiting the productivity of tef (Assefa et al., 2017). In Ethiopia, over 22 fungal pathogens have been known to cause disease on tef crop (Bekele, 1985). Of these, rust (Uromyces eragrostidis) and head smudge (Curvularia spp.) is the most economically important disease (Stewart and Yirgou, 1967; Tareke, 1981). Head smudge is the most destructive disease causing up to 50% yield loss in Ethiopia (Gorshkov and Mekonnen, 1979).

Earlier evidence in Ethiopia indicates that head smudge has been reported as the most important disease on tef starting from 1966 (Stewart and Yirgou, 1967). In most Ethiopian literatures (Stewart and Yirgou, 1967; Tareke, 1981; Amogne et al., 2001; Assefa et al., 2017; Bekele, 1985; Ketema, 1993; Ketema, 1997; Zerihun, 2011), the causative agent of head smudge is named as Helmithosporium miyakei. However, the taxonomic study conducted by (Sivanesan, 1987) the genes Helminthosporium that attacks graminicolous Spp. were segregated into Bipolaris, Curvularia, Drechslera, and Exserohilum. According to the taxonomic study conducted vr Sivanesan (1987) and based on the taxonomic and phylogenetic study of the pathogen, the causal agent of head smudge of tef is a Curvularia (Manamgoda et al. 2015).

Curvularia is a genus with saprophytes, endophytes and also pathogens of a wide range of plants and represents important pathogens of grass and staple crops (Marin-Felix et al. 2017). Curvularia is characterized by the formation of brown distoseptate conidia ((Fig. 2a), usually with its distinctive characteristic curvature. The curvature of the conidia is the main difference from the similar genus Bipolaris (Marin-Felix et al., 2020). The sexual morph is rarely found in nature and is difficult to induce in culture (Manamgoda et al., 2015). The asexual propagation of the conidia is common in nature. Members of Curvularia show different spreading and survival mechanisms, i.e. through infected soil, seeds or crop residues (Manamgoda et al., 2011; Kamil et al., 2020). Degete (2021) stated that infected seed and stubble are the main mechanisms of tef head smudge survival and spread. However, this requires confirmatory research. Head smudge disease initially shows a light blackening symptoms in the inflorescence tip, which progresses towards a branch of panicles with florets (Fig. 2c). When the relative humidity and temperature are favorable, the entire inflorescence turns dark brown in severe infection (Fig. 3b), forming a branch of panicles joined by the black mass of mycelium and the sooty of the pathogen.

Farmers in the western Amhara region have repeatedly complained about black symptoms on the panicle of tef and have asked for help to control them. A survey was conducted to identify the disease and to assess the distribution and intensity of the disease. According to (Zegeye et al., 2021) survey, the symptoms were caused by Curvularia spp., which caused head smudge with mean incidence of 20% and severity of 11.2% in tef producing areas in the region. While the head smudge becomes an economically important disease, especially in humid tef growing areas of Amhara region, resistant varieties have not been yet developed, nor any chemical control measurement have been evaluated. Hence, it is important to select and recommend effective fungicides for the management of the disease. Therefore, the experiment was conducted to identify effective fungicides against head smudge of tef and simultaneously to estimate possible effects of head smudge on tef seed quality.

Study area description

The experiment was conducted at hot spot areas of North Achefer and Dangila districts (each with 3 sites with 5 km distance between them). However, we were only able to collect data from one site at North Achefer in 2018, the other two sites were rejected. Dangila is located 80 km southwest of Bahir Dar. The district is situated in the north-western highlands with elevation generally vary between 1850 and 2350 meters above sea level (Gowing et al., 2020). The mean annual temperature ranges from 9.4°C to 25.2°C. The area receives 1600 mm total annual rainfall (Adane et al., 2020). Tef, maize, finger millet, potato, pulse and oil crops are the dominant crops frequently grown in the district. The soil types are highly variable in the district, with Nitisols being the dominant soil type. North Achefer is located 545 km northwest of Addis Ababa, the capital of Ethiopia and 102 km to the west of Bahir Dar. The elevation of the district varies from 1215 to 2691 meters above sea level. The area receives 1409 mm total annual rainfall. The study area has an average annual maximum and minimum temperature of 22.5°C and 16°C, respectively (Abebe and Sewnet, 2014). The major crops grown comprise maize, wheat, tef, finger millet and pulses. The soil type of these districts is predominantly characterised by Nitisols followed by clay soil.

Experimental materials, design and procedures

The study was carried out under natural infection conditions during the 2018 and 2019 main cropping seasons. Eight treatments, including seven commercial fungicides and untreated control, (Table 1) were laid out in a randomized complete block design (RCBD) with three replications. The plots were 12m2 (15 rows and 4 m in length). The space between rows, plots and replications were 0.2 m, 1 m, and 1.5 m, respectively. 15 kg ha^− 1 seed of a known variety Etsube was planted from early to mid-July. Recommended fertilizer rate of 46 kg ha^− 1 P₂O₅ was applied during planting. Additionally, recommended nitrogen rates of 46 and 28 kg ha^− 1 were applied at tillering on red and black soil, respectively. Weeds were managed by hand two times per season. Seed dressing fungicide was applied 24 hours before sowing. Foliar fungicides were sprayed started at post flowering, based on the factory recommendation rate (Table 1), with 300 l ha^− 1 of water using 1 litter capacity of ultra-low volume sprayer (ULV) at every 15 days interval for three times. During fungicide applications, plastic sheets were used to prevent inter-plot interference due to spray drift. The crop was protected from the infestation of shoot flies through the application of insecticide (lambda-cyhalotrin) based on the factory recommendation rate (0.5 l ha^− 1) with 250 l ha^− 1 of water using 20 litter capacity of knapsack sprayer in all experimental fields uniformly to avoid the yield losses due to insects.

The laboratory experiment was conducted at the plant pathology laboratory of Adet Agricultural Research Center. The eight treatments of the field experiment and the same cultivar were included in the laboratory experiment, which was organized in a completely randomized design (CRD) with four repetitions. Seeds were derived from the field experiment. The blotter method was used following International Rules for Seed Testing Association (ISTA, 2001). In this method, tef seeds were sown on layers of moistened blotters placed in 15cm diameter plastic petri dishes at the rate of 100 seeds per dish. Then the dishes were incubated under room temperature near fluorescent light with a 12 hr. cycle light and dark. The seeds were checked every day and the papers moistened with distilled water. After ten days of incubation, the seeds in petri dishes were examined for head smudge infection using a stereo binocular microscope. The fungi were identified by morphology using binocular compound microscope.

The disease was measured based on the incidence and severity. Incidence of tef head smudge was assessed by counting the number of diseased plants per total number of plants inspected per meter row and expressed as a percentage, according to the following equation. Plant Disease incidence (%) = $\:\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e}\text{d}\:\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}\:}{\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}\:\text{i}\text{n}\text{s}\text{p}\text{e}\text{c}\text{t}\text{e}\text{d}\:}\times\:\:100$

Disease severity was recorded from the panicles of 10 pre-tagged plants from the central rows of each plot diagonally, twice after the start of spraying, as a proportion of the area of plant tissue affected over the total area of plant tissue that has been infected by the disease (Madden et al., 2007).

Disease severity (%) = $\:\frac{\text{A}\text{r}\text{e}\text{a}\:\text{o}\text{f}\:\text{p}\text{l}\text{a}\text{n}\text{t}\:\text{t}\text{i}\text{s}\text{s}\text{u}\text{e}\:\text{a}\text{f}\text{f}\text{e}\text{c}\text{t}\text{e}\text{d}\:\:}{\:\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{p}\text{l}\text{a}\text{n}\text{t}\:\left(\text{p}\text{a}\text{n}\text{i}\text{c}\text{l}\text{e}\right)\:\text{a}\text{r}\text{e}\text{a}\:}\times\:\:100$

Relative efficacy of the each fungicides were calculated based on the formula used by Mayton et al. (2018):

$$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{e}\text{f}\text{f}\text{i}\text{c}\text{a}\text{c}\text{y}\:=\frac{\text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e}\:\text{s}\text{e}\text{v}\text{e}\text{r}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{p}\text{l}\text{o}\text{t}\:-\:\text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e}\:\text{s}\text{e}\text{v}\text{e}\text{r}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{p}\text{l}\text{o}\text{t}}{\text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e}\:\text{s}\text{e}\text{v}\text{e}\text{r}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{p}\text{l}\text{o}\text{t}}\times\:100$$

on the final day of the germination test, seedlings were grouped into (i) normal seedlings, (ii) abnormal seedlings (iii) ungerminated, and (iv) dead seeds. Finally, the germination percentage was calculated as follows;

Germination percentage(%)=$\:\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{n}\text{o}\text{r}\text{m}\text{a}\text{l}\:\text{s}\text{e}\text{e}\text{d}\text{l}\text{i}\text{n}\text{g}\text{s}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\:\text{s}\text{o}\text{w}\text{n}}\times\:$100

Maguire (1962) presented the germination rate for laboratory conditions to evaluate the seedling vigour. Following the author's protocol, the number of normal germinated seeds was recorded daily until there is no further germination. Maguire (1962) gave a generic mathematical expression for the method presented by Throneberry and Smith (1955) that permits one to obtain the measurement for any interval of time. The mathematical expression is given by

$$\:\text{G}\text{R}\:=\:\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{n}\text{o}\text{r}\text{m}\text{a}\text{l}\:\text{s}\text{e}\text{e}\text{d}\text{l}\text{i}\text{n}\text{g}\text{s}\:}{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{d}\text{a}\text{y}\text{’}\text{s}\:\text{t}\text{o}\:\text{t}\text{h}\text{e}\:\text{f}\text{i}\text{r}\text{s}\text{t}\:\text{c}\text{o}\text{u}\text{n}\text{t}}+\dots\:+\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{n}\text{o}\text{r}\text{m}\text{a}\text{l}\:\text{s}\text{e}\text{e}\text{d}\text{l}\text{i}\text{n}\text{g}\text{s}}{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{d}\text{a}\text{y}\text{’}\text{s}\:\text{t}\text{o}\:\text{t}\text{h}\text{e}\:\text{f}\text{i}\text{n}\text{a}\text{l}\:\text{c}\text{o}\text{u}\text{n}\text{t}}$$

For determination of seedling vigour, 10 seedlings per plot were selected randomly and their shoot and root length were measured in cm. The vigour of the seedlings was determined by the following formula:

Vigor Index = (Mean root length + Mean shoot length) x % seed germination

Grain yield of each plot was weighed by using sensitive balance after harvesting, drying, threshing, and winnowing. Finally, the yield obtained from each plot was converted in t ha^− 1 for statistical analysis.

The relative yield losses were expressed as the ratio of unprotected plot yield to the maximum attainable yield in protected plot with fungicides. Losses were calculated separately for each of the treatments with different levels of disease severity, based on the following formula (Savary and Willocquet, 2014).

$\:\text{R}\text{L}\:=\frac{\text{y}\text{i}\text{e}\text{l}\text{d}\:\text{o}\text{f}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{c}\text{t}\text{e}\text{d}\:\text{p}\text{l}\text{o}\text{t}\:-\:\text{y}\text{i}\text{e}\text{l}\text{d}\:\text{o}\text{f}\:\text{u}\text{n}\text{p}\text{r}\text{o}\text{t}\text{e}\text{c}\text{t}\text{e}\text{d}\:\text{p}\text{l}\text{o}\text{t}\:\:}{\text{y}\text{i}\text{e}\text{l}\text{d}\:\text{o}\text{f}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{c}\text{t}\text{e}\text{d}\:\text{p}\text{l}\text{o}\text{t}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:}\times\:10$ 0

Where; RL = relative yield loss

The cost and benefit of each treatment were analyzed partially, and the marginal rate of return was computed by considering the variable cost available in the respective treatment (CIMMYT, 1988). Yield and economic data were collected to compare the advantage of fungicide treatment. The grain yield obtained from each treatment was adjusted downwards by 10%. After harvesting, total gross benefit of the field was calculated from the grain yield of tef with the local market price (0.887 $ kg^− 1). The study utilized the 2020 average exchange rate of one US dollar to Ethiopian birr, which was 34.9505 ETB. Variable costs, such as the costs of fungicide and labor costs for fungicide application, were considered. The price of trifloxystrobin + tebuconazol and cymoxanil + copper oxychloride was similar 34.3 $ kg^− 1. While the price of all other fungicides were equal 28.6 $ kg^− 1. Also, the labor cost man-days was 2.6 $. Total variable cost (TVC) is the aggregate amount of all variable costs associated with the fungicide and labour costs. Net benefit (NB) is determined by subtracting all variable costs from the gross benefit as follows: NB = GB -TVC. The cost that varied for each treatment was ranked in order of ascending variable costs for dominance analysis. Any treatment that has net benefits that are less than or equal to those of a treatment with lower costs that vary is dominated (D). The marginal rate of return (MRR) was calculated for two non-dominated treatments (say 1 and 2) using the formula: [change in net benefit (NB2 − NB1)/change in TVC (TVC2 − TVC1)] × 100. Marginal rate of return provides the value of the benefit obtained per the amount of additional cost incurred percentage.

Meteorology data

The weather data of North Achefer were obtained from Bahirdar Meteorology Agency. While due to the absence of metrology station at Dangila, the data were not presented.

Data Analysis

The data generated from experiments were subjected to ANOVA test following the procedure described by Gomez and Gomez (1984) for one factor randomized complete block design (RCBD) and completely randomized design (CRD) using the PROC GLM procedure of SAS 9.4. Means were compared using the LSD test (α = 0.05). Prior to combined analysis over sites and years, normality and homogeneity tests were performed. The normality test was performed using the proc univariate procedure of SAS 9.4 statistical software. The homogeneity test (Levene's test) was conducted using SAS 9.4 statistical software's PROC GLM procedure with the MEANS statement (hovtest = levene option). Head smudge incidence and severity were log-transformed prior to analysis, as needed to improve the normality of the variables.

Incidence and severity

The statistical analysis indicated a significant (P < 0.0001) difference in incidence scores between fungicide treated and untreated plots at both locations and seasons (Tables SM 1 and 2). The highest disease incidence (97.8%) was observed in unsprayed plots at North Achefer, while at Dangila it was highest in thiamethoxam + metalaxyl + difenoconazole treated plots (Table 2). Moreover, no statistically significant variations in incidence scores were found between the unsprayed, cymoxanil + copper oxychloride, thiamethoxam + metalaxyl + difenoconazole, mancozeb + cymoxanil, and triadimefon treated plots in all locations, with the exception of triadimefon, which showed a significant difference at Dangila. The significantly lowest incidence score (< 76.6%) was recorded in propiconazole sprayed plots in both locations. Following propiconazole, the lowest incidence scores (<80.8% and <84.2%) were recorded from tebuconazole and tebuconazole plus trifloxystrobin treated plots. Also, the difference between tebuconazole and tebuconazole plus trifloxystrobin was not significant in both locations. However, at North Achefer, the incidence scores recoded from tebuconazole plus trifloxystrobin (84.2%) were not significantly different from triadimefon (90.9%) (Table 2).

The effect of fungicides in reducing disease severity was significant (P < 0.0001) (Tables SM 1 and 2). Some of the foliar spray fungicides significantly decreased the level of disease severity in comparison with the control and other ineffective fungicides (Table 2). Maximum level of protection (93.4 and 94.5 %) with reduced severity (5.1 and 4.6%) was provided by the spraying of propiconazole at Dangila and North Achefer, respectively (Table 2). Also, disease control provided by the application of tebuconazole plus trifloxystrobin and tebuconazole was not significantly different from propiconazole. The efficacy of these treatments compared to the control plot was 93.4 and 91.5% at Dangila and 88.1 and 91.7% at North Achefer, respectively (Table 2). Besides, triadimefon was moderately effective with 85.9% and 75.6% relative efficacy at Dangila and North Achefer, respectively. Higher infection development resulted in a lower level of disease control provided by cymoxanil + copper oxychloride and mancozeb + cymoxanil treated plots (Table 2). Also planting treated tef seed with thiamethoxam + metalaxyl + difenoconazole failed to reduce disease severity, which was statistically similar to the unsprayed plot. In both locations, a maximum level of disease infection was recorded on tef plant panicles in untreated plots before crop maturity, with mean values of 77.3 and 83.3% of panicle area covered with head smudge at Dangila and North Achefer, respectively.

Among the two consecutive seasons, weather conditions during 2019 were highly favorable for head smudge infection development, resulting in the early onset of the disease, causing a high level of disease incidence and severity in untreated plots at North Achefer. In 2019, there was relatively extended rainfall and highest temperature than 2018 at North Achefer (Fig. 1). Comparatively, during the 2018 cropping season, early onset of the disease with maximum incidence and severity was reported at Dangila. In general, head smudge was severely occurred at North Achefer than Dangila (Table 2). This is due to the proximity of North Achefer's to Lake Tana as well as the presence of high temperatures (Fig. 1), both of which contribute to increased humidity. Daget (2021) stated that disease intensity increases near water bodies (rivers) and in fields bordered by shade trees. Even though the disease pressure was varied across locations and seasons, all the treatments displayed similar trends to either successfully or unsuccessfully minimize disease infection. The use of demethylation inhibitors (DMI) fungicides proved to be more efficient in the control of head smudge diseases when applied in the heading stages (Daget, 2023). Several studies reported the higher efficacy of tebuconazole propiconazole and triadimefon based fungicides against many diseases caused by Thecaphora frezii, Puccinia spp., Septoria spp., and powdery mildew on different crops (Paredes et al., 2020; Osborne and Stein, 2009; Phatik et al., 2011; Pramod and Dwivedi, 2007; Fondevilla and Rubiales, 2012). DMI fungicides are among the most common broad-spectrum economically important agricultural chemicals that are widely used on wheat, barley, and orchard fruits (Buchenauer, 1987) to control powdery mildew, rust, brown rot, and leaf spot disease (Woods et al., 2005). They are absorbed and translocated in the plant, where they act preventively (before infection) or curatively (in the presence of symptoms) by affecting germ tube and appressoria formation and/or mycelial growth (Pontzen and Scheinpflug, 1989). Triazole fungicides exhibit their antifungal activity by inhibiting fungal ergosterol biosynthesis, which is a fundamental component of the fungal cell plasma membrane (Brent, 1995), and regulatory compounds (e.g., inhibiting steroid hormone synthesis) (Bloch, 1983). The absence of ergosterol and the increase of intermediate compounds alter fungal membrane integrity as well as cell morphology, which inhibits fungal growth (Becher et al., 2010).

Grain Yield

Grain yield was significantly (P < 0.0001) influenced by fungicide treatment in both seasons and locations (Tables SM 1 and 2). At Dangila, over the two seasons, the use of trifloxystrobin + tebuconazol resulted in highest mean yield (610.5 kg ha^-1) (Table 2). Also, the seasonally combined yield (582.4kg ha^-1) produced by tebuconazol were not significantly different from trifloxystrobin + tebuconazol. Correspondingly, the mean yield (533.4 kg ha^-1) produced by propiconazole treated plots was not significantly different from tebuconazole treated plots (Table 2). The remaining fungicides in order of superiority were triadimefon followed by mancozeb + cymoxanil which were significantly superior over the control (Table 2). Spraying cymoxanil + copper oxychloride and treating the seed with thiamethoxam + metalaxyl + difenoconazole did not provide significant yield improvement from the untreated plot (250.7 kg ha^-1) (Table 2). Generally, yields obtained at Dangila were below the average due to severe infestation of shoot fly in both seasons but similar trends were observed across all treatments.

Similarly, in the study conducted at North Achefer, the use of fungicides significantly (P < 0.0001) positively influenced the tef yield (Table SM 2). In the area, the highest mean yield (1538.0kg ha^-1) was produced by tebuconazole treatment. However, the yields obtained from experimental plots treated by trifloxystrobin +tebuconazol (1493.0 kg ha^-1) and propiconazole (1482.5kg ha^-1) were not significantly different from tebuconazole (Table 2). The lowest yield (555.1 kg ha^-1) was produced by unsprayed plot followed by application of seed dressing chemical thiamethoxam + metalaxyl + difenoconazole and foliar spraying of cymoxanil + copper oxychloride with 588.8and 641.7 kg ha^-1mean yield, respectively (Table 2). Interestingly, triadimefon also showed promising results, producing an average yield of 1342.2 kg ha^-1 (Table 2). Experimental plots that received mancozeb + cymoxanil produce an average yield of 760.0 kg ha^-1 (Table 2).

Generally, the application of tebuconazole, propiconazole, and triadimefon based foliar fungicides significantly increased yield in comparison with the control and other ineffective fungicides. Depending on the environment and the type of DMI fungicide used, the sprayed plots produced a mean yield of 190.9 kg ha^-1 to 982.9 kg ha^-1 more than the unsprayed plots. Even though there is insignificant variation between triazole fungicides, the maximum yield was obtained from experimental plots treated by tebuconazole followed by propiconazole and triadimefon. This is because, over time, triadimefon accumulates at the leaf margins, leaving other parts of the leaf more open to infection. But tebuconazole is active over the whole leaf for a longer period, giving more sustained control (Fondevilla and Rubiales, 2012).

However, foliar application of broad spectrum fungicides with contact, protective and curative action (mancozeb + cymoxanil and cymoxanil+ copper oxychloride) were less effective to increasing yield. Rather, cyanoacetamide fungicide cymoxanil with mancozeb and copper oxychloride is very effective against a wide range of downy mildew and late blight causing plant pathogens in potato, tomato, and melons (Knowles, 1998; Joshi, 2003). The result obtained from a seed treatment with thiamethoxam + metalaxyl + difenoconazole showed a similar result with untreated control. Similarly, the review made by Bekele (1985) showed that no significant results have been obtained from seed dressing experiments conducted at Bako Research Station against tef diseases. This is mainly because the duration of protection provided by seed treatment chemicals is short due to the relatively small amount of chemical applied to the seed, dilution of the chemical as the plant grows, and breakdown of the chemical (Paulsrud et al., 2001).

Yield loss

Yield losses were generally higher in 2019 than in 2018 at both Dangila and North Achefer. The overall combined data revealed that the loss of yield for the test variety Etsube was 62.0% compared with the best fungicide sprayed plots (Table 3). The relative yield loss (63.9%) at North Achefer was higher than the 58.9% loss at Dangila (Table 3). Of all the treatments, the higher loss % were obtained from the unprotected plots. On the average, the lowest relative yield loss (< 15.9 %) was observed from the DMI fungicides. On the other hand, mean relative yield loss obtained from mancozeb + cymoxanil, cymoxanil + copper oxychloride, and thiamethoxam + metalaxyl + difenoconazole were between 46.8% and 59.5% (Table 3). Depending upon locational and seasonal variations, the yield loss on the unsprayed plot were 40 to 70.5% compared with the best fungicide sprayed plots. Additionally, grain harvested from head smudge affected fields was of low quality due to a mixture of diseased or rotted blackened grains, which varied depending on environmental and disease management conditions (Fig. 4 T1 to T8). This substantially reduces market acceptance of the grain. Previous studies showed that the effect of head smudge on tef was estimated up to 50% yield loss (Gorshkov and Mekonnen, 1979) but the complete destruction of tef fields was observed around Ghimbi (CADU, 1969). Earlier evidence in Ethiopia showed that tef head smudge causes serious damage to both the yield and quality of tef grains in humid and warm areas of Southwest Ethiopia (Yirgou, 1967). Recently, Daget (2023) also reported 55.8 % tef yield loss in West Arsi Zone.

Loss in germination capacity

Highly significant differences were observed between treatments (P < 0.0001) in germinated and/or non-germinated seeds (Table 4). Germination of seeds declined from 76.4% to 43.8% as the percentage of rotted seeds increased from 18.7 % to 45.4 % depending on fungicide treatments applied at field condition (Table 4). Tef seeds had significantly (P < 005) higher germination in propiconazole (76.4%) and tebuconazole (73.8%) treated plots than untreated (50.2%) and other fungicide treatments (Table 4). Seed germination percentage produced from trifloxystrobin + tebuconazole (63.8%) and triadimefon (62.6%) treated plots was moderate. Around half of the seeds were failed to germinate in seeds produced from cymoxanil + copper oxychloride (47.9%), mancozeb + cymoxanil (48.3%) and thiamethoxam + metalaxyl + difenoconazole (52.4 %) treated plots (Table 4). Seed rotting due to head smudge infections detected from cymoxanil + copper oxychloride, mancozeb + cymoxanil and thiamethoxam + metalaxyl + difenoconazole treated plots were 41.7%, 43.1% and 45.4%, respectively (Table 4). Similar trends were observed in abnormally germinated seedlings. Treatments that showed highest germination (propiconazole, tebuconazole, trifloxystrobin + tebuconazole and triadimefon) resulted low abnormal seedlings (1.3%, 1.4%, 1.9% and 1.6%) (Table 4). However, less seed germination and more abnormal seedlings with the highest germination failure were observed in seeds produced with less effective fungicides and untreated. Soomro et al., (2020) found significant differences in seed germination and seedling health between treated and untreated seeds.

The result of laboratory data clearly showed that best protected plots under field conditions had the highest percent germination, while heavily infected plots resulted in lower percent germination. As the disease level on the tef panicle increased, seed rotting due to head smudge, abnormal seedling development, and overall germination failure increased, while germinated seeds decreased. This loss of seed quality could be associated with head smudge infection. In support of this different authors reported that Curvularia spp., detected in untreated Amaranth seeds significantly reduced percent germination (Alam et al., 2014; Islam, 2005; Hamim et al., 2014). Sinha and Prasad (1981) reported less seed germination of mung bean as a result of Alternaria infection.

Effect on germination rate

The germination rate of the tef seeds produced under different fungicide treatments was highly significant at P < 0.0001 (Table 4). The significantly fastest rate of seed germination was obtained from propiconazole (18.9) and tebuconazole (18.0) treated plots as compared to all other treatments (Table 4). A good result produced from trifloxystrobin + tebuconazole (15.6) and triadimefon (15.1) treated plots and they were statistically similar. A significantly slower rate of germination was obtained from thiamethoxam + metalaxyl + difenoconazole (10.6) followed by mancozeb + cymoxanil (12.1) and cymoxanil + copper oxychloride (11.7) treated plots (Table 4). However, no significant differences in germination rate were observed between the last four treatments and the untreated control (12.6 days).

Generally, the germination rate of tef seedlings was higher in protected than unprotected plots. According to Ranal and Santana (2006) "treatments that showed high values of germination rate should have higher seedling vigour".

Effect on seedling growth

No significant differences were observed in seedling shoot length after field treatments with different fungicides. However, the results of different treatments had a significant effect on seedling root length, height, and vigour index. (Table 5). The root length of seedlings derived from propiconazole (3.5 cm), tebuconazole (3.7cm), and trifloxystrobin + tebuconazole (3.7cm) treated plots were significantly bigger than cymoxanil + copper oxychloride (3.0cm), mancozeb + cymoxanil (3.2cm) and from thiamethoxam + metalaxyl + difenoconazole (3.1cm) treated plots (Table 5). In harmony to this Soomro et al. (2020) reported healthy seedlings of rapeseed due to fungicide treatment showed proper root and shoot system but seedlings raised from fungal spore inoculated seeds showed reduced root and shoot system. Unexpectedly, similar results of 3.4 cm were observed between triadimefon treated plots and untreated. Similar results were observed in seedling height. This is because seedling height is the result of the sum of shoot length and root length. Likewise, the vigor index data (Table 5) showed that propiconazole, tebuconazole, and trifloxystrobin + tebuconazole treated plots resulted in most vigor seedlings than untreated and other fungicides.

Partial budget analysis

The partial budget analysis indicated that some fungicide treatments resulted in high net benefit and marginal rate of return (Table 6 and Table 7). At Dangila, over the two seasons, the applications of tebuconazole, tebuconazole + trifloxistrobin, and propiconazole brig the greater net benefit (414.2, 402.3, and 375.1 $, respectively) (Table 6). While triademefon provides a moderate net benefit 258.9$. However, the net benefit obtained from cymoxanil + copper oxychloride, thiamethoxam + metalaxyl + difenoconazole, and mancozeb + cymoxanil (182.6, 174.6, and 114.0$, respectively) were below the untreated (200.1$) (Table 6). On similar way, at North Achefer the highest net benefit was provided through the applications of tebuconazole (1177.1$), followed by propiconazole (1132.8$), and tebuconazole + trifloxistrobin (1106.8$) (Table 7). The application of triadimefon provides net benefit of 977.9$. On the other hand, the net benefit obtained from the application of mancozeb + cymoxanil and thiamethoxam + metalaxyl + difenoconazole was lower than the untreated (443.1$) (Table 7). The highest marginal rate of return was achieved with the application of tebuconazole, with rates of 2326.5% and 7256.3% at Dangila and North Achefer, respectively (Table 6 and Table 7). Tebuconazole + trifloxystrobin also showed MRR of 79.4% at Dangila. While the other treatments were dominated. Overall, the partial budget analysis suggest that fungicides in a triazole family (sterol demethylation inhibitors) are the most economically beneficial options for controlling tef head smudge disease.

Findings in this study revealed that head smudge could severely reduce the yield of tef in both seed and/or grain quality and quantity. Based on the 2 years and 10 sites data, the average yield loss caused by the disease was 62% on test variety Etsube compared with best protected plots. Also, seed quality parameters germination percentage, germination rate, seedling root length, seedling height, and vigor index were influenced to the same extent as yield, significant differences occurred between severely and less infected plots. Depending upon treatments applied, 18.7 to 45.4% germination failure due to seed rotting were observed. However, the applications of fungicides such as propiconazole, trifloxystrobin + tebuconazole and tebuconazol were reduce germination failure. In the same way, the application of those fungicides were significantly decrease disease severity and increased yield as compared to the untreated. Thus, it is recommended farmers and seed multipliers to spray fungicides trifloxystrobin + tebuconazol, tebuconazole, and propiconazole interchangeably based on market availability, to minimize risks of head smudge in grain and seeds of tef.

Competing of interest

The authors declare that they have no competing of interest.

Author Contribution

Mequanint Andualema wrote the manuscript and provided the data for field and laboratory experiments. He is also responsible for data analysis and interpretation. Walellign Zegey contributes to the work's conception or design. Gebremariem Asaye and Bogale Nigir help to critically revise it for important intellectual content. Girmay Dires and Melkamu Birhanie help with field data collection and fungicide application, Aderajew Mihiretie help to critically revise it for important intellectual content.

Acknowledgment

The authors would like to thank Amhara Agricultural Research Institute (ARARI) for providing research funding.

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Table 1. Description of different fungicides used for foliar spray to manage tef head smudge at Dangila and North Achefer during the 2018 and 2019 cropping seasons

Treatments	Trade name	Active ingredient	Modes of application	Rate as factory
1	Nativo SC 250	Trifloxystrobin 100 gm/lt + Tebuconazole 200 gm/lt	Foliar	0.75 kg ha^-1
2	Natura 250 EW	Tebuconazole	Foliar	0.5 l ha^-
2	Tilt 250 EC	Propiconazole	Foliar	0.5l ha^-
4	Noble 25 WP	Triadimefon	Foliar	1 kg ha^-1
5	More 720 WP	Mancozeb + cymoxanil	Foliar	2 kg ha^-1
6	Curzate R WP	Cymoxanil+copper oxychloride	Foliar	0.3 kg ha^-1
7	Apron star 42 WS	(Thiamethoxam 20% + metalaxyl - 20% + difenoconazole 2%)	Seed dressing	0.375 kg ha^-1
8	Untreated

Table 2 Effect of different fungicide applications on tef yield and head smudge incidence (%) and severity (%) at Dangila and North Achefer during the 2018 and 2019 cropping seasons

No.	Fungicide	Dangila				North Achefer
No.	Fungicide	GY (kg ha^-1	Inc.%	Sev.%	RE.%	GY(kg ha^-1	Inc.%	Sev.%	RE.%
1	Trifloxystrobin + tebuconazol	610.5 ^a	82.2(1.91) ^c	5.1(0.67) ^d	93.4	1493.0^a	84.2(1.92) ^bc	9.9(0.82) ^c	88.1
2	Tebuconazole	582.4 ^ab	80.8(1.90) ^c	6.6(0.72) ^d	91.5	1538.0 ^a	80.8(1.90) ^c	6.9(0.75) ^cd	91.7
2	Propiconazole	533.4^b	76.6(1.87) ^d	5.1(0.66) ^d	93.4	1482.5^a	75.0(1.86) ^d	4.6(0.61) ^d	94.5
4	Triadimefon	441.6^c	86.9(1.94) ^b	10.9(0.93) ^c	85.9	1342.2^b	90.9(1.95) ^ab	20.3(1.17) ^b	75.6
5	Mancozeb + cymoxanil	367.5 ^d	95.1(1.98) ^a	51.2(1.68) ^b	33.8	760.0 ^c	92.8(1.96) ^a	69.4(1.80) ^a	16.7
6	Cymoxanil+copper oxychloride	274.9 ^e	97.1(1.99) ^a	66.3(1.79) ^a	14.2	641.7^d	97.5(1.99) ^a	75.9(1.84) ^a	8.9
7	Thiamethoxam + metalaxyl + difenoconazole	269.3 ^e	97.3(1.99) ^a	70.7(1.81) ^a	8.5	588.8 ^d	95.0(1.98) ^a	77.4(1.85) ^a	7.1
8	Control	250.7 ^e	96.7(1.98) ^a	77.3(1.87) ^a	0	555.1^d	97.8(1.99) ^a	83.3(1.90) ^a	0
	LSD	65.12	0.02	0.09		117.87	0.04	0.16
	P-value	<.0001	<.0001	<.0001		<.0001	<.0001	<.0001
	CV %	23.6	1.9	11.2		13.8	2.3	14.8

GY = Grain yield, Inc. = incidence, Sev. = Severity, RE = Relative efficacy, kg = kilograms, ha = hectare, CV. = coefficient of variation; Means within a column followed by the same letter are not significantly different at their respective level of significance; Means in the parentheses are transformed means

Table 3. Relative Yield loss (%) compared to superior fungicide at each season and location

No.	Fungicides	Dangila			North Achefer			Over all mean
No.	Fungicides	2018	2019	combined	2018	2019	combined	Over all mean
1	Trifloxystrobin + tebuconazol	0.0	0.0	0.0	2.0	4.4	2.9	0.8
2	Tebuconazole	2.7	6.4	4.6	4.8	0.0	0.0	0.0
2	Propiconazole	5.0	20.0	12.6	0.0	5.8	3.6	4.9
4	Triadimefon	25.3	30.0	27.7	9.8	14.6	12.7	15.9
5	Mancozeb + cymoxanil	32.8	46.6	39.8	24.5	58.0	50.6	46.8
6	Cymoxanil+copper oxychloride	52.4	57.5	55.0	24.8	67.5	58.3	56.8
7	Thiamethoxam +metalaxyl +difenoconazole	49.0	62.6	55.9	22.1	72.5	61.7	59.5
8	Control(unprotected)	50.6	67.1	58.9	40.3	70.5	63.9	62.0

Table 4. Combined mean of germination rate, germination percent, abnormal seedling, dead seeds and rotted seeds

No.	Fungicides	GR	NG (%)	ABGS (%)	TG (%)	DS (%)	RS (%)	GF (%)
1	Trifloxystrobin + tebuconazol	15.6^b	63.8^b	1.9^c	65.7^b	11.2	23.1^cd	34.3^c
2	Tebuconazole	18.0^a	73.8^a	1.4^c	75.1^a	4.1	20.8^d	24.9^d
2	Propiconazole	18.9^a	76.4^a	1.3^c	77.7^a	3.6	18.7^d	22.3^d
4	Triadimefon	15.1^b	62.6^b	1.6^c	64.2^b	7.0	28.8^c	35.8^c
5	Mancozeb + cymoxanil	12.1^cd	47.4^c	4.3^ab	51.7^cd	5.2	43.1^ab	48.3^ab
6	Cymoxanil+copper oxychloride	11.7^cd	46.6^c	5.6^a	52.1^cd	6.2	41.7^ab	47.9^ab
7	Thiamethoxam + metalaxyl + difenoconazole	10.6^d	43.8^c	3.8^b	47.6^d	7.1	45.4^a	52.4^a
8	Untreated control	12.6^c	50.2^c	5.0^ab	55.3^c	6.3	38.5^b	44.8^b
	LSD	1.7	7.2	1.7	7.1	-	6.8	7.1
	P-value	<.0001	<.0001	<.0001	<.0001	0.1659	<.0001	<.0001
	CV	17.4	17.8	36.9	16.6	49.7	29.9	26.2

LSD = Least significant difference; CV = Coefficient of variation; GR = Germination rate; NG = normally germinated seedling; ABGS = abnormally germinated seedlings; TG = totally germinated seeds; DS = Dead seeds; RS = Rotted seeds; GF = germination failurity, Means within a column followed by the same letter are not significantly different at their respective level of significance.

Table 5. Combined mean of shoot length, root length, seedling height and seedling vigour index

No.	Fungicide	Shoot length (cm)	Root length (cm)	Seedling height (cm)	Seedling vigour index
1	Trifloxystrobin + tebuconazol	2.0	3.7^a	5.8^a	375.8^ab
2	Tebuconazole	2.0	3.7^a	5.7^a	431.2^a
2	Propiconazole	2.0	3.5^ab	5.5^ab	422.3^a
4	Triadimefon	1.9	3.4^bc	5.2^bc	335.3^bc
5	Mancozeb + cymoxanil	2.0	3.2^bc	5.1^bc	257.6^d
6	Cymoxanil+copper oxychloride	1.8	3.0^c	4.9^c	248.7^d
7	Thiamethoxam + metalaxyl + difenoconazole	1.9	3.1^c	4.9^c	229.5^d
8	Untreated control	1.9	3.4^bc	5.2^bc	281.0^dc
	LSD	-	0.3	0.35	59.5
	P-value	0.1970	<.0001	<.0001	<.0001
	CV%	13.7	13.2	9.6	26.3

Cm = centimeter, LSD = Least significant difference; CV = Coefficient of variation; Means within a column followed by the same letter are not significantly different at their respective level of significance.

Table 6. Partial budget analysis of the effect fungicide application on tef head smudge management at Dangila during the 2018 and 2019 cropping seasons

No.	Items	Average Yield(kg ha-1 )	Adjusted yield (10%) kg ha-1	Gross benefit ($ ha-1)	Total variable cost ($ ha-1 )	Net benefit ($ ha-1)	MRR%
8	Untreated control	250.7	225.6	200.1	0.0	200.1	-
6	Cymoxanil+copper oxychloride	274.9	247.4	219.4	36.8	182.6	D
7	Thiamethoxam + metalaxyl +difenoconazole	269.3	242.4	215.0	40.3	174.6	D
2	Tebuconazole	582.4	524.2	464.9	50.6	414.2	2326.5
3	Propiconazole	533.4	480.1	425.8	50.7	375.1	D
1	Trifloxystrobin + tebuconazol	610.5	549.5	487.3	85.0	402.3	79.4
4	Triadimefon	441.6	397.4	352.5	93.6	258.9	D
5	Mancozeb + cymoxanil	367.5	330.8	293.4	179.4	114.0	D

MRR = marginal rate of return; kg = kilograms, ETB = Ethiopian birr, ha = hectare, D= dominated

Table 7. Partial budget analysis of the effect fungicide application on tef head smudge management at North Achefer during the 2018 and 2019 cropping seasons

No.	Items	Average Yield(kg ha-1)	Adjusted yield (10%) kg ha-1	Gross benefit($ ha-1)	Total variable cost ($ ha-1)	Net benefit ($ ha-1)	MRR%
8	Untreated control	555.1	499.6	443.1	0.0	443.1	-
6	Cymoxanil+copper oxychloride	641.7	577.5	512.3	36.8	475.4	87.7
7	Thiamethoxam + metalaxyl +difenoconazole	588.8	529.9	470.0	40.3	429.7	D
2	Tebuconazole	1538.0	1384.2	1227.7	50.6	1177.1	7256.3
3	Propiconazole	1482.5	1334.3	1183.4	50.7	1132.8	D
1	Trifloxystrobin + tebuconazol	1493.0	1343.7	1191.8	85.0	1106.8	D
4	Triadimefon	1342.2	1208.0	1071.4	93.6	977.9	D
5	Mancozeb + cymoxanil	759.9	683.9	606.6	179.4	427.2	D

MRR = marginal rate of return; kg = kilograms, ETB = Ethiopian birr, ha = hectare, D= dominated

No competing interests reported.

ANOVA.docx

Fungicide evaluation against head smudge (Curvularia spp.) and its impact on yield and germination of tef (Eragrostis tef Zucc.)

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Abstract

Figures

Introduction

Materials and methods

Study area description

Experimental materials, design and procedures

Meteorology data

Data Analysis

Results and Discussion

Conclusions and Recommendations

Declarations

Competing of interest

Author Contribution

Acknowledgment

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1