Antifungal Activity of Organic Manure Extractsagainst Exerohilum Turcicum, Causal Agent of Helminthosporiasis of Maize (Zea mays L.)

Helminthosporiosis of maize (Zea mays L.) is one of the cryptogamic diseases of maize, causing enormous yield losses. Chemical fertilizers and fungicides are most often used for soil fertilization and disease control. Inorder to develop ecologically acceptable alternative methods, several results were reported on the antifungal potential of compost juice or tea. The overall aim of this study was to evaluate the antifungal activity of four types of organic manure extracts against Exerohilum turcicum, the causal agent of maize helminthosporiosis. Two batches of aqueous organic manure extracts (sterilized and non-sterilized) at concentrations C1= 0.375 μL/mL; C2= 0.75 μL/mL; C3= 1.5 μL/mL and C4= 3 μL/mL were used. These organic manures consisted of extracts from beef manure (EBM), household waste (EHW), pig manure (EPM) and poultry manure (EPTM). The chemical fungicide (FONGI) Ridomil Gold plus 66WP was used as a negative control. Radial growth inhibition rates of the pathogen as a function of extract concentrations were evaluated. The results showed that unsterilized extracts had a high inhibitory power compared with sterilized extracts. Non-sterilized household manure extracts showed the highest inhibition rate (IR (EHWM)= 77.32%) at concentration C4= 3 μL/mL, and very low inhibition (IR (EHWMS)= 0.34%) at concentration C1= 0.375 μL/mL in sterilized household waste manure extracts, compared with the negative control, which had an inhibition rate of 100%. Principal component analysis (PCA) showed a wide variability in the inhibitory power of the extracts. In addition, high pathogen inhibition rates were obtained at high concentrations (C3= 1.5 μL/mL and C4= 3 μL/mL). Fresh organic manure extracts are said to contain substances that inhibit the mycelial growth of Exerohilum turcicum. This antifungal potential of organic manures can be integrated into biological control methods against Helminthosporiasis of maize. 

Introduction

Maize (Zea mays L.) is one of the most common cereal crops grown in Africa. A large proportion of the population in sub-Saharan Africa depends on maize as their main staple food crop (Badu-Aprakuet al., 2021). It is one of the most versatile crops, characterized by great adaptability to varied agro-ecological and climatic conditions (Mohammedet al., 2023). Maize is used to feed people and animals, and in a number of economic circuits. After rice and wheat, maize is the world’s most important cereal crop (Atri, 2019). In Cameroon, it is grown alone or in association with other crops by small-scale farmers and is mainly intended for self-consumption. Out of an estimated demand of 2.8 million tonnes, annual national maize production is around 2.3 million tonnes, a deficit of 500 thousand tonnes (Anonymous, 2020). This low production is for the most part linked to biotic constraints. According to Boulenouaret al. (2011), fungi cause around 10,000 different plant diseases. Among the most dreaded fungal diseases affecting maize production in Cameroon there is helminthosporiasis, also known as leaf blight. It is characterized by numerous lesions on the leaves, causing grain yield losses ranging from 28% to 91% (Rajeshwar Redyet al., 2021). To deal with this threat, synthetic fungicides are the means of control generally used. However, according to Avilés and Maria (2011), the intensive use of physical/chemical biocides generates a microbiological vacuum in the soil/substrate that makes it more susceptible to re-infestation by pathogens, increases disease incidence and, in some cases, enhances fungal resistance. Indeed, although chemical pesticides are effective, their environmental and health risks are cause for concern (Bouchtaouiet al., 2024). A number of ecologically acceptable studies have been carried out on the potential of organic fertilizers to suppress certain plant diseases (Kutnjem, 2020; Larbi, 2006; Ngakouet al., 2014; Ndja’a, 2015; Ndongo, 2019 and Ndongoet al., 2022). This work has shown both fertilizing and phytosanitary benefits for certain crops, including maize. Indeed, soil microbiota play a key role in crop protection by enhancing the natural suppression capacity of soil pathogens (De Corato, 2020). However, few studies have clarified the effects of abiotic and biotic components of compost on the soil microbial community and the growth of these crops (Luoet al., 2022). The aim of this work was therefore to test in-vitro the antifungal activity of four types of organic manure extracts on the development of E. turcicum, the causal agent of Helminthosporiasis of maize.

Material and method

Pathogen Isolation and Identification

Maize leaves showing symptoms of helminthosporiasis were collected from Akonolinga, chief town of Nyong and Mfoumou department, Centre region (N 03° 47^′ 32.2^′′ and E 012° 15^′ 48^′′). Leaves were washed with tap water, surface sterilized with a 5% sodium hypochlorite solution for three minutes, rinsed with distilled water and then air-dried. The necrotic surfaces were scraped with a scalpel and spread between slide and coverslip to observe the conidia under a light microscope. Once the presence of conidia was confirmed, the diseased parts of the leaves were cut into small 3 mm-5 mm pieces and placed on PDA medium in solidified Petri dishes, sealed with parafilm. After six days of growth in Petri dishes incubated at a temperature of 23°C ± 2°C. Successive subcultures were performed on new PDA medium until pure E. turcicum isolates were obtained.

Obtaining Different Organic Fertilizers and Preparing Aqueous Extracts

Obtaining Organic Manures

Organic manures were obtained from beef, pig and poultry dung collected at the beef market and from pig and poultry farms in Yaoundé. Household refuse manure was purchased in composted form from the Centre International de la Promotion de la Récupération (CIPRE) in the same city. The collected dung was air-dried for seven days, then mixed with Tithonia diversifolia and Chromoleana odorata (Fig.1A). The heaped mixture was turned every 14 days for six months (the time required for ripening), sometimes preceded by watering with well water or rainwater, depending on availability. After ripening, each heap was sieved using a 5 mm diameter sieve and placed in bags for drying in a greenhouse (Fig.1C). After six days, each sample was passed through a second, finer sieve 2 mm in diameter in the laboratory and placed in sterile plastic bags with a mass capacity of 50 g (Fig.1D).

Fig. 1. Some preparatory stages of the organic manures used. A: Fresh animal dung +Tithonia diversifolia; B: Pile of manure in decomposition phase; C: Pile in maturation phase; D: Bagged samples for various analyses.

Preparation of the Different Extracts from the Manures Obtained

Extracts were prepared according to the modified protocol of Agbodjato (2017). Two batches were prepared: a sterilized batch and a non-sterilized batch. A total of eight extract samples were used in the experiment. Indeed, 10 g of each type of fertilizer were taken, weighed and then diluted in 90 ml of distilled water to obtain a 10-1 dilution of stock solution (Fig. 2). This procedure was repeated for the second batch of extracts intended for sterilization. The stock solutions from the first batch, considered as non-sterilized extracts, were designated as: Extract of Beef Manure (EBM), Extract of Household Waste (EHWM), Extract of Pigs (EPM) and Extract of Poultry (EPTM). Each solution was stirred for 4 min and left for six days in the fridge at a temperature of 4°C. The above extraction procedure was repeated to obtain the second batch for sterilization. These were represented by Sterilized Beef Manure (EBMS), Sterilized Household Waste (EHWMS), Sterilized Pigs (EPMS) and Sterilized Poultry (EPTMS). After this extraction time, the various solutions were then pre-filtered using muslin and filtered through a 0.2 µm membrane filter (Kutnjem, 2020). The second batch was autoclaved at 121°C for 20 min, then cooled before use. The positive control received 30 ml of PDA culture medium and the negative control (FONGI) received a mixture of 0.5 ml fungicide (Ridomil Gold plus 66WP) and 19.5 ml PDA culture medium, for a final volume of 30 ml. The aim of the sterilized batch was to demonstrate the beneficial effect of the heat-sensitive or heat-stable microorganisms contained in the extracts, and to gain a better understanding of their involvement in the process of inhibiting the mycelial growth of the pathogen under study. Four concentrations C1 = 0.375 µL/mL; C2 = 0.75 µL/mL; C3 = 1.5 µL/mL and C4 = 3 µL/ mL of each stock solution were added to four volumes of PDA culture medium of 29.625 mL; 29.25 mL; 28.5mL and 27mL respectively, giving a final volume of 30 mL. Positive controls were not amended with extract. After homogenization, the mixture was poured at 40°C into 90 mm-diameter Petri dishes in a hood, around a flame. The petri dishes were kept in the hood until the medium had solidified (Dogaet al., 2022). The test was repeated three times for each batch.

Fig. 2. Different aqueous extracts of organic manures used. A: Non-sterilized extracts in the extraction phase; B: Sterilized extracts; EBM: Extract of beef manure; EHWM: Extract of household waste manure; EPM: Extract of pig manure; EPTM: Extract of poultry manure.

Evaluation of the Efficacy of Aqueous Organic Manure Extracts on the Development of E. turcicum

To assess the efficacy of manure extracts on the radial growth of the pathogen under study, fragments of pure isolates 6 mm in diameter were taken with a cookie cutter from the growth front of the fungus. They were then placed in the geometric center of the Petri dish on solidified PDA culture medium. The seeded petri dishes were sealed with adhesive film and placed in an oven at 25°C ± 2°C for eight days. Radial growth of the fungus was assessed at 2-day intervals using two perpendicular axes traced at the base of each Petri dish, converging in the middle of the explant. The different diameters were calculated using the formula proposed by Singhet al. (1993).

$$\begin{array}{r}\text{D}=\frac{\text{D}1+\text{D}2}{2}-\text{D}0\end{array}$$

where D0 is the explant diameter, D1 and D2 are the culture diameters measured in the two perpendicular directions (Fig. 3).

Fig. 3. Measuring mycelial growth in Petri dishes on PDA medium.

For each volume corresponding to a given concentration, 4 Petri dishes were seeded. Controls were colonies growing on PDA media unamended with extract. Each treatment was repeated three times. The efficacy of each extract was determined from the inhibition rate calculated using mean diameter measurements according to the formula used by Konéet al. (2009); Kumaret al. (2007) and Narayanasamy (2013):

$$\begin{array}{r}IR\left(\mathrm{\%}\right)=(Dto\left(mm\right)-Dxi\left(mm\right))/Dto\left(mm\right)\times 100\end{array}$$

where IR (%) is inhibition rate, Dto is the mean diameter of the control batch and Dxi the mean diameter of batches in the presence of the extract.

Dto is mean radial growth value for control treatments; Dxi is mean radial growth value for trial treatments. IR is inhibition rate.

Statistical Analysis

The one-way statistical analysis of variance followed by the principal component analysis associated with this study was carried out on the Rcmdr (R commander) graphical interface of the R software Version 4.4.1. and on XLSTAT. The Shipiro-wilk and Levene normality tests for homogeneity were carried out at the 5% threshold. The simultaneous comparison of the confidence intervals by the Tukey test made it possible to evaluate the significant differences between the inhibition percentages. As for the histograms, they were drawn up on Excel 2016.

Results

Pathogen Isolation and Identification

Petri dishes containing the pathogen were colonized over time by E. turcicum mycelial filaments until they were completely filled. The morphological characteristics of conidia, conidiophores and hyphae were identical to those often found in E. turcicum. Microscopic observations showed that conidia in all Petri dishes containing the isolate were curved and elliptical in shape, with a fusiform tip. In fact, the mycelia of the colonies were black in color and developed in a circular relief. They also featured grey aerial hyphae. Conidia from the recovered fungi were pale brown, long and fusiform with 2–13 transverse septa (Fig. 4).

Fig. 4. Microscopic observations of the E. turcicum culture. A: Pure strain; B: Colony of conidia and hyphae; C: Septate conidia (x40).

Comparative Effect of Four Types of Unsterilized Organic Manure Extracts on E. turcicum Mycelial Growth

The results of this experiment showed that the aqueous extracts of household refuse (EHWM) and beef manure (EBM) significantly reduced the radial growth of the pathogen (p < 5%) (Figs. 5 and 6). In fact, mycelial growth of E. turcicum was significantly inhibited in petri dishes treated with EHWM and EBM, compared with mycelial growth of E. turcicum in petri dishes not treated with extracts, whose colonization by E. turcium mycelium was complete after six days of incubation (Fig. 5). None of the extracts compared with the chemical fungicide inhibited 100% of the pathogen’s radial growth. However, extracts containing household waste manure showed the highest inhibition rate (IR (EHWM) = 77.32%) at concentration C4 = 3 µL/mL, while extracts containing poultry manure showed the lowest inhibition rate (IR (EPTM) = 5.02%) at this concentration (Figs. 5 and 6).

Fig. 5. In-vitro inhibition of E. turcicum mycelial growth by fresh aqueous manure extracts of different concentrations. EBM: Extract from beef manure; EHWM: Extract from house waste manure; EPM: Extract from pig manure; EPTM: Extract from poultry manure; FONGI: chemical Fongicide IR: inhibition rate; C: Concentration.

Fig. 6. Inhibition rate of unsterilized aqueous organic manure extracts as a function of concentration. EBM: Extract from beef manure; EHWM: Extract from Household; EPM: Extract from pig manure; EPTM: Extract from poultry manure; FONGI: chemical Fongicide; IR: inhibition rate; Conc: Concentration.

Comparative Effect of the Four Types of Sterilized Organic Manure Extracts on Mycelial Growth of E. turcicum

Evaluation of the effect of sterilized organic manure extracts on the growth of the fungus studied revealed a significant loss (p < 5%) of their inhibitory power at all concentrations. In-vitro tests showed almost normal mycelial growth of the pathogen in the presence of these types of extracts (Fig. 7). Near-zero inhibition rates were recorded with EHWMS and EPMS. The lowest inhibitory power was obtained by sterilized household waste manure extracts (IR = 0.34%) at concentration C1 = 0.375 µL/mL compared with the control. The highest inhibition percentages were obtained by EPMS sterilized beef manure extract (IR = 12.72%) at concentration C4 = 3 µL/mL (Fig. 8).

Fig. 7. In-vitro inhibition test of H. turcicum radial growth by sterilized manure extracts at different concentrations. EBMS: sterilized Extract from beef manure; EHWMS: sterilized Extract from household manure; EPMS: sterilized Extract from pig manure; EPTMS: sterilized Extract from poultry manure; FONGI: chemical Fongicide IR: inhibition rate; C: Concentration.

Fig. 8. Inhibition rates of aqueous extracts of sterilized organic manures as a function of concentration. EBMS: sterilized Extract from beef manure; EHWMS: sterilized Extract from household manure; EPMS: sterilized Extract from pig manure; EPTMS: sterilized Extract from poultry manure; FONGI: chemical Fongicide IR: inhibition rate; Con: Concentration. C1 = 0.375 µL/mL; C2 = 0.75 µL/mL; C3 = 1.5 µL/mL; C4 = 3 µL/mL.

Analysis of Quantitative Variables

In order to examine the relationships between the various inhibition rates and concentrations, a principal component analysis was carried out. The results obtained are presented in the correlation circle and the graph of observations in Fig. 9. The correlation circle shows that the first dimension of the PCA (F1) represents 70.30% of the original information, while the second dimension accounts for only 16.47%. There was a positive correlation between inhibition rates (IR EPMS, IR EHWM, IR EPTMS, IR EPM, IR EBMS and IR EHWMS) and a negative one between IR EPTM and IR EBM. However, the IR EPMS and IR EHWMS variables were not related. The graph of observations showed that concentrations C3 = 1.5 µL/mL and C4 = 3 µL/mL were characterized by high variability in inhibition rates, in contrast to concentrations C1 = 0.375 µL/mL and C2 = 0.75 µL/mL.

Fig. 9. Principal component analysis of the variables studied. IR: Inhibition rates for unsterilized and sterilized extracts. C: Concentrations (C1 = 0.375 µL/mL; C2 = 0.75 µL/mL; C3 = 1.5 µL/mL; C4 = 3 µL/mL).

Discussion

Morphological Characterization of the Exserohilum turcicum Isolate, the Causal Agent of Helminthosporiosis of Maize

The pure strain resulting from the morphological study obtained after isolation was identified as Exserohilum turcicum, the pathogenic agent of Helminthosporiasis of maize. Microscopic observation at ×40 magnification showed spindle-shaped, elongated and septate conidia, and the presence of a protruding hilum at the end of the conidia was noted, constituting the unique character of the E. turcicum spore. In addition, the color of the mycelium was brown and developed more rapidly (Bashiret al., 2017).

Inhibition of E. turcicum Mycelial Growth by Manure Extracts

Effect of Unsterilized Extracts on Mycelial Growth

The reduction in mycelial growth ranged from 5.02% to 77.32%. This variability in inhibitory effect depended essentially on the nature of each extract. Indeed, the suppressive power of organic fertilizers on plant diseases depended on the input composition of each type. Such variation was reported in the work of Ringeret al. (1997) when comparing the effect of composts from different types of manure against Pitium ultimum and Rhizoctonia solani. Indeed, these authors noted that cattle manure composts proved more effective against P. ultimum than horse manure composts, the most effective being chicken manure compost. In the present study, the highest inhibition rates were obtained by EBM, EHWM and EPM, while EPTM recorded the lowest rates. Of all the extracts used in this experiment, EHWM was the most effective at C4 concentration (3 µL/mL), with an inhibition percentage equal to 77.32%. These results are similar to those obtained by Kutnjem (2020) on the same issue. Indeed, working on the effect of household waste composts on the radial growth of Helminthosporium turcicum, the causal agent of maize helminthosporiasis, this author obtained a reduction in the pathogen’s growth with an inhibition rate equal to 76.92% at concentration C3 (2 µL/mL). These two results are therefore justified by the presence in this substrate of a specific microbial community whose role is to produce bioactive substances that inhibit the pathogen’s mycelial growth. This justification is supported by the work of Fuchs (2003), who studied the ability of composts to protect watercress plants against Pythium ultimum, the causal agent of damping-off. According to this author, a compost of high microbiological quality has the power to protect plants against disease, whereas a less microbiologically active compost does not. Finally, the degree of mineralization of organic manures has often been cited by some authors as a credible criterion for assessing the phytosanitary power of their extracts.

Effect of Sterilized Extracts on Growth

Sterilization of the extracts made it possible to verify whether or not the high inhibition rates observed previously were due to the microorganisms present in these samples. The results showed that all four sterilized aqueous extracts had a very low inhibitory power in some cases, and even zero in others. Radial pathogen growth inhibition rates ranged from 0.34% to 12.72%. EHWMS recorded the lowest inhibition rate (IR.EHWMS = 0.34%) at C1 concentration, while the highest inhibition rate was observed on EBMS (IREBMS = 12.72%) at C4. As before, the variation in these results could be explained by the uneven distribution of microbiota, which depends on the nature of each type of extract. The low inhibition rates are attributable to the destruction of microorganisms antagonistic to the pathogen responsible for maize helminthosporiasis. This microbial population would therefore be predominantly made up of thermophilic actinomycetes rather than mesophilic fungi (Soreet al., 2021). According to Trillas-Gay (1986) the suppressive effect is reduced or even eliminated by heating the compost to 60°C for at least four hours or by irradiating it with gamma rays. This result does not corroborate the results obtained in a similar study by Kutnjem (2020) who, when evaluating the inhibitory power of sterilized extracts of household waste composts, observed a significant reduction in the radial growth of the Helminthosporium turcicum strain, with a percentage inhibition of around 95.89% at concentration C3 (2 mL aqueous compost extract). The highest concentration (C4) appears to have induced the highest inhibition rate. This result may be explained by the fact that a high percentage of beneficial microorganisms are eradicated, leaving only the physico-chemical properties of the compost to emerge. The results of Fuchs (2009) on the effect of compost on fertility and phytopathogens showed that heat treatment of active compost destroys its active microflora, resulting in the loss of its suppressive power. El Kinanyet al. (2017) make a similar point when they suggest that this reduction in mycelium growth in the 40% concentration of sterilized compost extract may be due to certain chemical compounds developed during the composting process and remaining after the sterilization stage, or to certain thermostable extracellular metabolites.

Analysis of Quantitative Variables

Principal component analysis between the different inhibition rates obtained and the concentrations showed that increasing the concentration contributed to a greater diversity of inhibitory power, resulting in the best inhibition rates of manures against Exerohilum turcicum.

Conclusion

In-vitro examination of the effect of organic manure extracts on Exserohilium turcicum, the causal agent of helminthosporiasis of maize, showed a wide variability in the inhibitory potential of these extracts on the growth of the pathogen. This variability was linked to the nature of each substrate, which took into account the basic inputs. Unsterilized extracts of the various organic fertilizer substrates significantly inhibited pathogen mycelial growth, in contrast to sterilized extracts. High inhibition rates were obtained with unsterilized organic manure extracts (IR (EHWM) = 77.32%) at concentration C4 = 3 µL/mL and very low (IR (EHWMS) = 0.34%) at concentration C1 = 0, 375 µL/mL with sterilized organic manure extracts, compared with the negative control which had an inhibition rate of 100%. The fresh organic manure extracts contain substances that inhibit the mycelial growth of Exerohilum turcicum. This antifungal potential of organic manures can be integrated into control methods.