Understanding the environmental and herbicide response of Lasiodiplodia theobromae and Bipolaris bicolor isolated from infected Eleusine indica

Abstract:

Background:  In a prior study, Lasiodiplodia theobromae (Pat.) Griffiths and Maubl. and Bipolaris bicolor (Mitra) Shoemaker., were found to suppress the growth of Eleusine indica (L.) Gaertn, but limited information exists on their response to environmental factors and herbicides for integrated E. indica control.

Objective:  This study aimed to determine the tolerance levels of L. theobromae and B. bicolor to pH, temperature, photoperiod, relative humidity, and herbicides.

Methods:  The mycelia and conidia of L. theobromae and B. bicolor were exposed to a range of environmental conditions and herbicides in a controlled setting, including different levels of temperature (25, 30, 35 ⁰C), pH (4, 6, 8, 10), photoperiod (24 hours of darkness; alternating 12 hours of blue light followed by 12 hours of darkness; and alternating cycles of 6 hours of blue light and 6 hours of darkness), and relative humidity (75, 85, 95%). Herbicides such as topramezone, diuron, oxyfluorfen, and imazethapyr were applied at their recommended rates. Conidial germination was assessed by counting after an 18-hour incubation using a haemocytometer, while the diameter of mycelium growth was measured after 3 days of incubation, except for the herbicide effects evaluation, which were extended over 7 days.

Results:  Laboratory assays demonstrated that L. theobromae exhibited higher conidial germination of 85-95% and superior mycelial growth under varied pH, temperature, and photoperiod conditions compared to B. bicolor. Lasiodioplodia theobromae's mycelia remained unaffected by herbicides at full labelled rates, but they inhibited the conidial germination of the fungus. For B. bicolor, the conidia were not affected by imazethapyr and topramezone, but its mycelial growth was reduced by imazethapyr, oxyfluorfen, and diuron.

Conclusions:  These results indicate that the mycelia of L. theobromae presents a more favorable option for tank mixing with test herbicides, offering potential for the formulation of an integrated control strategy against E. indica.

Keywords:  Lasiodiplodia theobromae ; Bipolaris bicolor ; Environmental Factors; Herbicides

1. Introduction

Eleusine indica (L.) Gaertn, an annual grass weed, thrives in diverse agricultural regions across Malaysia, encompassing oil palm plantations, orchards, and vegetable cultivations (Chuah, Sahid, 2010). However, the indiscriminate use of various herbicides has fostered resistance in this weed against multiple herbicide groups (Dilipkumar et al., 2020a). Consequently, numerous studies have sought to mitigate herbicide usage and forestall the emergence of herbicide-resistant E. indica by employing a blend of chemical and physical methods (Amirul et al., 2019a; 2019b; Chuah, Lim, 2021a; 2021b; Chuah et al., 2018; Dilipkumar et al., 2020b). Despite these efforts, the integration of biological and chemical strategies for controlling E. indica remains underexplored.

In crop fields, weeds are vulnerable to fungal colonization, with certain fungi capable of producing mycotoxins under specific conditions (Giorni et al., 2019). Several fungi, including Lasiodiplodia theobromae (Pat.) Griffiths and Maubl. and Bipolaris bicolor (Mitra) Shoemaker, have demonstrated efficacy in suppressing E. indica growth (Fakri et al., 2023). The colonization and mycotoxin production in fungi are influenced by eco-physiological variables such as temperature and relative humidity (Lahouar et al., 2015). Furthermore, climate change can yield diverse impacts on fungal colonization and mycotoxin production, ranging from inhibition to stimulation or no effect at all (Samapundo et al., 2007; Akbar et al., 2016; Luck et al., 2011).

Concurrently, herbicides interact multifariously with plants and microorganisms, altering the plant-pathogen relationship while targeting weeds (Duke et al., 2007). Changes induced by herbicides to the host plant's structure and defenses may heighten susceptibility to infection (Ruuskanen et al., 2023). Understanding herbicide effects on fungal pathogens is pivotal for formulating integrated strategies for weed control (Charudattan, 2001). When herbicides are applied sequentially or as tank mixtures, it is imperative to ensure that those with synergistic potential do not impede the spores and mycelium of fungal pathogens (El-Sayed, 2005). For example, a recent study by Zhang et al. (2022) demonstrated the efficacy of combining Bipolaris eleusines Alcorn and Shivas conidia with bensulfuron or penoxsulam in controlling barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.], monochoria [Monochoria vaginalis (Burm.f.) Presl C. ex Kunth.], and small-flower umbrella sedge [Cyperus difformis (L.)] in rice fields. Hoagland et al. (2018) found that formulations containing technical-grade glyphosate and Myrothecium verrucaria (Alb. and Schwein.) Ditmar ex Fr., mycelia, effectively managed palmer amaranth [Amaranthus palmeri (S. Wats.)] by significantly reducing plant height by over 50% and fresh weight by 90%.

Hence, the ease of cultivating fungi in laboratory settings and their high compatibility with herbicides position them as promising candidates for integrated weed management. However, unlocking their potential necessitates a comprehensive understanding of their reproductive characteristics influenced by environmental factors, crucial for large-scale production and adaptability in field applications, and their compatibility with herbicides for effective weed suppression. Thus, this study aimed to investigate the effects of various environmental factors, including temperature, photoperiod, pH, humidity, and herbicides such as topramezone, imazethapyr, oxyfluorfen, and diuron, on the mycelial growth and conidial germination of L. theobromae and B. bicolor. These herbicides were selected because E. indica has not evolved resistance to them (Heap, 2023). The data obtained from this research could furnish valuable insights into the fungi's tolerance to environmental conditions and their interactions with herbicides, thereby playing a pivotal role in the integrated control of E. indica through both biological and chemical approaches.

2. Material and Methods

2.1 Mycelium preparation

The pure cultures of L. theobromae and B. bicolor were obtained from Fakri et al. (2023), Laboratory of Agrotechnology, Complex Star, Universiti Teknologi MARA, Arau, Perlis, Malaysia. Lasiodiplodia theobromae culture was sup-cultured onto fresh potato dextrose agar (PDA) (Merck KGaA, Darmstadt, Germany) medium in a 6-cm-diameter Petri dish using a 5-mm-diameter cork borer and incubated at 27 ⁰C under darkness for 7 days in an incubator. The same procedure was repeated for B. bicolor for 14 days. Five-mm-diameter of mycelial discs taken from the margin of the colony was used for supsequent experiments into temperature, photoperiod, pH, humidity, and herbicide effects.

2.2 Conidia preparation

For harvesting conidia of L. theobromae, approximately 10 mL of sterilized potato dextrose broth (PDB) (Merck KGaA, Darmstadt, Germany) was poured onto a 7-day-old colony culture grown on PDA medium, and conidia were scrapped off using a sterilized glass rod. The same procedure was repeated for B. bicolor using a 14-day-old colony culture. The conidial suspension of the fungus was filtered through two layers of sterilized muslin cloth, and then adjusted to approximately 1.0 × 10⁸ conidia/mL using a haemocytometer and transferred into a 50 mL universal bottle for supsequent experiments into temperature and photoperiod effects.

2.3 Temperature effect

The 5-mm diameter mycelial discs of each fungus were placed on PDA medium and incubated at temperatures of 25, 30 and 35 ⁰C, respectively, under darkness. Diameter of mycelium growth was recorded daily for three days (Siva et al., 2017).

The conidia suspension in universal bottle was incubated at 25, 30 and 35 ⁰C for 18 hours under darkness. Conidial germination was counted in the haemocytometer under a compound microscope where conidia was considered germinate when a germ tube was longer than the conidia diameter (Benslim et al., 2016). More than 500 conidia were counted for each treatment. The conidial germination was expressed in percentage as follows:

$$\text{Conidial germination}=\frac{\text{Number of germinated conidia}}{\text{Total number of conidia}}\times 100$$

2.4 Photoperiod effect

The 5-mm diameter mycelial discs of each fungus were placed on PDA medium and exposed to oscillate cycles of 24 hours dark; 12 hours blue light and 12 hours dark; and alternate 6 hours blue light and 6 hours dark in a growth chamber maintained at 27 ⁰C. Diameter of mycelium growth was recorded daily for three days as described before. Meanwhile, the conidial suspension in universal bottle was exposed to different photoperiods as described above at 27 ⁰C for 18 hours. Conidial germination was counted using the haemocytometer. Conidial germination was expressed in percentage as described in Section 2.3 (Siva et al., 2017).

2.5 Humidity effect

The 5-mm diameter mycelial discs of each fungus were placed on PDA medium and exposed to different relative humidity of 75, 85 and 95%, respectively, and incubated at 27 ⁰C under darkness in a closed container sized 34 cm (L) × 24.5 cm (H) × 19.5 cm (W). The relative humidity of 75, 85 and 95% was simulated using saturated salt solution of sodium chloride (NaCl) (HmbG^® Chemicals, Malaysia), potassium chloride (KCl) (QReC™, Thailand) and potassium sulphate (K₂SO₄) (SRL Chem, India), respectively (Hong et al., 2005) and confirmed using digital humidity meter. The diameter of mycelium growth was recorded daily for three days after incubation as described in Section 2.3.

2.6 pH effect

The pH levels of the PDB and PDA were adjusted accordingly using a digital pH meter by adding 1.0 N hydrochloric acid (HCl) and 1.0 N sodium hydroxide (NaOH) to attain pH 4.0, 6.0, 8.0 and 10.0. The PDA medium was sterilized and poured into Petri dish and allowed to solidify. The 5-mm-diameter mycelial disc of each fungus was placed on the centre of Petri dish and incubated at 27 ⁰C under darkness. Diameter of the fungal mycelium was recorded daily for three days as described in Section 2.3. 10 mL of sterilized PDB with pH 4.0, 6.0, 8.0 and 10, were respectively; poured onto pre-prepared fungal culture grown on PDA medium and the conidia were scrapped off. Then, the conidial suspension was filtered, transferred into the universal bottle, and adjusted to 1.0 × 10⁸ conidia/mL using the haemocytometer and incubated at 27⁰C for 18 hours under darkness. Conidial germination was counted and expressed in percentage as described in Section 2.3 (Siva et al., 2017).

2.7 Herbicide effect

Commercial grades of four herbicides examined in this experiment were topramezone, diuron, oxyfluorfen, and imazethapyr (Table 1). The herbicides were diluted with sterilized PDA or PDB to attain respective manufacturer-recommended rates. The PDA without herbicide incorporation was used as non-treated control. The herbicide containing PDA was allowed to solidify, followed by the placement of a pre-prepared 5-mm-diameter mycelial disc at the center of the agar medium in a Petri dish. Subsequently, the dish was incubated at 27⁰C for 7 days in darkness. (Bussaman et al., 2012). Diameter of mycelium growth was recorded and expressed as percentage of respective non-treated control as follows:

$$\text{Mycelium growth}=\frac{\text{Diameter of treated mycelium}}{\text{Diammeter of non treated mycelium}}\times 100$$

Table 1
Characteristics of selected commercial grade herbicides.

Active ingredient	Trade name	Mode of action*	Formulation
Topramezone	CLIO ™ SC	Inhibits the enzyme 4-Hydroxy-Phenyl-Pyruvat-dioxygenase (4-HPPD).	Suspension concentrate (SC)
Diuron	ANCOM DIURON F42	Inhibits photosynthesis at photosystem II.	Suspension concentrate (SC)
Oxyfluorfen	BOXY ZEENEX	Inhibition of the enzyme protoporphyrinogen oxidase (PPO or Protox).	Emulsifiable concentrate (EC)
Imazethapyr	IMAZ 5.2SL	Inhibition of branched amino acid production by inhibition of the enzyme acetolactate synthase (ALS) or acetohydroxy acid synthase (AHAS).	Soluble concentrate (SL)

The herbicide-treated PDB at respective recommended rate was poured onto fungal culture grown on the pre-prepared PDA medium and the conidia were scrapped off (Celar, Kos, 2016). The conidial suspension was filtered with muslin cloth, transferred into the universal bottle, and incubated at 27 ⁰C for 48 hours under darkness. The PDB without adding herbicide was used as non-treated control. Conidial germination was counted in the haemocytometer under a compound microscope. The conidial germination was expressed as percentage of respective non-treated control as follows:

$$\text{Conidial germination}=\frac{\text{Number of germinated herbicide treated conidia}}{\text{Number of germinated non treated conidia}}\times 100$$

3. Experimental design and Statistical analysis

The experiments on effects of temperature, photoperiods and humidity were laid out as split plot in a completely randomized design with five to six replications and repeated twice where main plot is temperature level, photoperiod, or humidity level whereas supplot is type of fungal pathogen. The data were checked for the normality and homogeneity of variance being supjected to ANOVA, followed by Tukey's test to compare means at 5% level of significance. The experiments on the effects of pH and herbicide were laid out as factorial in a completely randomized design with five to six replications where factor one is type of fungal pathogen, whereas factor two is pH level or herbicide type. The data were checked for the normality and homogeneity of variance before being supjected to two-way ANOVA, followed by Tukey's test to compare means at 5% level of significance. All statistical procedures were conducted using JMP software version 14.

4. Result and Discussion

4.1 Environmental effects on conidial germination

4.1.1 Effects of temperature

The effects of temperature on the conidial germination rates of B. bicolor and L. theobromae are illustrated in Figure 1a. Considering the conidial germination rate, a notable interaction was observed between the type of fungus and the temperature factor. These two fungi were able to germinate, with L. theobromae having a high conidial germination rate of 86 to 90% at temperatures between 25 and 35 ⁰C. In comparison, B. bicolor conidial germination rate was just 45% to 47% (Figure 1a). The conidial germination rates of B. bicolor were not affected significantly (p>0.05) by the rise in temperature. By contrast, when the temperature increased, the conidial germination rates of L. theobromae increased marginally (p<0.05). Unlike B. bicolor isolates from Malaysia, Xiao et al. (2021) found that the optimum temperature for conidial germination of B. bicolor taken from a tea garden in Zhejiang, China, was 25 ⁰C, whereas the conidial germination rate was significantly lower at 35 ⁰C.

Figure 1
Conidial germination rate of Lasiodiplodia theobromae (Bipolaris bicolor () and ) under different environmental factors. Each datum is the average ± SD of six replicates. At least 500 conidia were counted for each value. A. Conidial germination rate at temperatures of 25, 30 and 35 ⁰C for 18 hours under darkness. B. Conidial germination rate at pH 4, 6, 8 and 10 at 27 ⁰C for 18 hours under darkness. C. Conidial germination rate at 27 ⁰C for 24 hours under darkness (), alternating 6-h blue light () and alternating12-h blue light (). Means followed by a different lowercase letter have a significant difference at the 5% significance level

4.1.2 Effects of pH

The effects of pH on the conidial germination rates of B. bicolor and L. theobromae are depicted in Figure 1b. A significant interaction was observed between the conidial germination rate of the fungus type and the pH factor. The pH had a supstantial effect (p<0.05) on the conidial germination rate of L. theobromae, with the conidial germination rate increasing from 84% at pH 4 to 91% at pH 10. Any increase in acidity or alkalinity had no effect on the germination rate of B. bicolor conidia, which constantly had a rate of 30 to 32% (Figure 1b). In contrast to present findings, Xiao et al. (2021) reported that the optimum pH for conidial germination of B. bicolor was 7 whereas high acidity and alkalify at pH 4 and pH 10, respectively could reduce the conidial germination rate supstantially

4.1.3 Effects of photoperiod

The effects of photoperiod on the conidial germination rates of B. bicolor and L. theobromae are outlined in Figure 1c. There was a significant interaction between the conidial germination rate of fungus type and photoperiod factor. The effect of light exposure duration on the conidial germination rate of the test fungus was not consistent. It was discovered that L. theobromae treated to 6 hours of alternating blue light had a greater conidial germination rate than those exposed to 24 hours of darkness and 12 hours of alternating blue light. On the other hand, B. bicolor's conidial germination rate could be reduced by 24 hours of darkness. Unlike B. bicolor isolates from Malaysia, conidia of B. bicolor from China exhibited the highest germination at fluorescent light (Xiao et al., 2021). Moreover, L. theobromae exhibited 75 to 85% conidial germination rate either at alternating photoperiod or complete darkness.

The present findings on the optimal temperature, pH and photoperiod for conidial germination of B. bicolor do not align with the results of Xiao et al (2021). These discrepancies could be attributable to diverse geographic regions, potentially reflecting adaptation of the fungal pathogen (Kumar et al., 2010). Besides, in addition to displaying high pathogenicity against target weeds, a mycoherbicide must be highly adaptable to environmental conditions such as temperature, pH and photoperiod (Hassan et al., 2021). The formulation of choice for majority of effective mycoherbicide is conidia. A fungal mycoherbicide agent must also have a rapid sporulation rate (Verma et al., 2021). In this study, conidia of L. theobromae can be harvested from 7-day-old cultures, whereas B. bicolor requires 14-day-old cultures to produce similar conidial density, indicating that L. theobromae has advantages over B. bicolor for mycoherbicide development. The current investigation also revealed that B. bicolor exhibited a low conidial germination rate of 20 to 50% whereas L. theobromae had a higher conidial germination rate ranging from 75 to 95% across various levels of temperature, pH and photoperiod. These results indicated that L. theobromae outperformed B. bicolor and was more adaptable to a wide range of environmental conditions, implying its promise as a better mycoherbicide candidate.

4.2 Environmental effects on mycelial growth

4.2.1 Effects of temperature

Figure 2a presents the effects of temperature on mycelial growth of B. bicolor and L. theobromae. Intriguingly, the mycelial growth patterns of the test fungus differed from the conidial germination rate. An interaction between the fungus type and temperature factor influenced the mycelium growth significantly. Mycelia of L. theobromae and B. bicolor grew more slowly at 35 ⁰C, decreasing in diameter from 5.5 to 4.5 cm and 2 to 1 cm, respectively, demonstrating that the rise in temperature from 25 to 35 ⁰C could inhibit the growth of both fungi. Thus, mycelia of L. theobromae and B. bicolor, were clearly not tolerant to temperature fluctuations.

Figure 2
Mycelial growth of Lasiodiplodia theobromae (Bipolaris bicolor (), and ) under different environmental factors. Each datum is the average ± SD of five replicates. A. Mycelial growth at temperatures of 25, 30 and 35 ⁰C for 24 hours under darkness. B. Mycelial growth at pH 4, 6, 8 and 10 for 24 hours at 27 ⁰C under darkness. C. Mycelial growth at 27 ⁰C for 24 hours under darkness (), alternating 6-h blue light () and alternating12-h blue light (). D. Main effect of relative humidity on mycelial growth at 27 ⁰C for 24 hours averaged across fungus type under darkness. Means followed by a different lowercase letter have a significant difference at the 5% significance level

Channakeshave and Pankaja (2018) observed that the optimal temperature for the radial growth of Bipolaris oryzae (Breda de Haan) Shoemaker isolated from paddy was 30 ⁰C, with lower and higher temperatures inhibiting the growth of the fungus. Arshad et al. (2013) also noticed that the B. oryzae performed optimally between 25 and 35 °C. Saha et al. (2008) demonstrated that L. theobromae could develop at temperatures between 8 and 36 ⁰C, with optimum growth occurring at 28 ⁰C. Alam et al. (2001) also observed that L. theobromae grew at temperatures ranging from 10 to 40 ^oC, with the ideal temperature range falling between 25 and 30 ⁰C. In accordance with the findings of Alam et al. (2001), the optimum temperature for mycelia development of L. theobromae was found to be between 25 and 30 ⁰C in current research.

4.2.2 Effects of pH

Figure 2b depicts the effects of pH on mycelial growth of B. bicolor and L. theobromae. Similar to temperature effect, the mycelial growth patterns of the test fungus varied from the conidial germination rate. The mycelium growth was significantly influenced by the interaction between the fungus type and pH factor. The mycelial growth of B. bicolor was marginally enhanced when the pH was increased from 4 to 10. By contrast, Channakeshave and Pankaja (2018) revealed that the optimal pH range for B. oryzae was between 6 and 7. On the other hand, the mycelial growth of L. theobromae was optimum at pH 6 but significantly hindered at pH 8 and 10. Saha et al. (2008) also found that L. theobromae could thrive in a pH range from 3.5 to 8.0 but the ideal pH range was between 5.5 and 6.5. Dheepa et al. (2018) discovered that L. theobromae grew most rapidly at pH 7.

4.2.3 Effects of photoperiod

Figure 2c illustrates the effects of photoperiod on mycelial growth of B. bicolor and L. theobromae. The mycelial growth patterns of the test fungus differed from the conidial germination rate. The interaction between the fungus type and photoperiod factor had a significant impact on the mycelium growth. Mycelial growth of L. theobromae was greater in the dark than under alternating blue light, indicating that darkness was more conducive to the growth of the fungus. However, Saha et al. (2008) found that light had no significant influence on the mycelial growth of L. theobromae under full light, complete darkness, and alternate 12-hour light treatments. Dheepa et al. (2018) also showed that there was no correlation between light regimes and the mycelial development of L. theobromae. The photoperiod had no marked influence (p>0.05) on the mycelial growth of B. bicolor. The findings are not in accordance with results of Channakeshave and Pankaja (2018) who found that B. oryzae exhibited greater mycelial growth when exposed to complete darkness as opposed to alternating light.

4.2.4 Effects of humidity

Figure 2d presents the effects of humidity on mycelial growth of B. bicolor and L. theobromae. The interaction between mycelium growth of fungal type and humidity effect was not significant. Since the main effect of humidity on the mycelial growth of the test fungi was not statistically significant (p>0.05), only the main effect of humidity level was presented. The data demonstrated that the test fungi were tolerant of humidity changes between 75% and 95%. However, mycelia of B. bicolor and L. theobromae grew better with humidity levels ranging from 85% to 95%.

The presence and activity of mycelium play a significant role in influencing pathogenic fungus infections in plants. Following the initial infection by the conidia of pathogenic fungi in weeds, the mycelium serves to perpetuate and spread the infection. As conidia mature into mycelium, they execute the invasion process. Therefore, the survival of mycelium in various environmental conditions such as temperature, photoperiod, pH, and humidity is crucial, similar to the survival of conidia. Understanding the impact of environmental factors on the growth of fungal pathogens’ mycelium is also essential for optimizing manufacturing processes. The greater mycelium growth observed in L. theobromae across a wide range of environmental conditions suggests its superiority over B. bicolor, thus positioning L. theobromae as a highly promising candidate for mycoherbicide applications.

4.3 Herbicide effects on conidial germination and mycelial growth

Figure 3 shows the effects of imazethapyr, topramezone, oxyfluorfen and diuron on conidial germination rate and mycelial growth of B. bicolor and L. theobromae. There was a significant interaction between fungus type and herbicide treatment on conidial germination rate (Figure 3a). It was noted that L. theobromae and B. bicolor, all reacted differently to herbicide treatments. L. theobromae was extremely susceptible to all test herbicides because the fungus only gave a 15–21% conidial germination rate following herbicide treatments. On the other hand, B. bicolor was tolerant to both imazethapyr and topramezone treatment; nevertheless, oxyfluorfen and diuron treatment significantly reduced the conidial germination of this fungus by 30–35%. In contrast to the trend in conidial germination rate, herbicide treatments had only a little (0–6%) inhibitory effect on L. theobromae mycelial growth (Figure 3b). Surprisingly, topramezone promoted mycelial growth of B. bicolor, although other test herbicides reduced the fungus’ growth by 50–60%.

Figure 3
Responses of Lasiodiplodia theobromae and Bipolaris bicolor after herbicide treatment of imazethapyr (), topramezone (), oxyfluorfen () and diuron () at respective commercial recommended rates. A. Conidial germination rate at 27 ⁰C for 48 hours under darkness after herbicide treatment. Each datum is the average ± SD of six replicates. At least 500 conidia were counted for each value. B. Mycelium growth at 27 ⁰C for seven days under darkness after herbicide treatment. Each datum is the average ± SD of five replicates. Means followed by a different lowercase letter have a significant difference at the 5% significance level

Based on the findings of this research, only topramezone had no detrimental impact on B. bicolor, but imazethapyr, oxyfluorfen, and diuron hindered conidial germination and mycelial growth. Likewise, Nemoto et al. (2002) demonstrated that glyphosate suppressed the mycelial growth of Bipolaris euphorbiae (Y. Nisik.) by 97%, regardless of dosage applied. Imazethapyr was also proven to diminish the biomass of the fungus. By contrast, bentazon had no effect on the fungus, but lactofen, a protoporyhyrinogen oxidase (PROTOX) inhibitor like oxyfluorfen, reduced mycelium growth by 85%. As revealed by Sanyal and Shrestha (2008), fungi possessed the same PROTOX enzyme targeted by the herbicide in plants, therefore it was not surprising that oxyfluorfen had a fungistatic impact on B. bicolor. Since B. bicolor was not affected by topramezone, this result suggested that the fungus was likely capable of degrading the herbicide. Although a diuron-degrading endophyte fungus isolated from sugarcane root has been reported to degrade diuron by up to 99% in three days under optimal conditions (Wang et al., 2017), B. bicolor isolated from the leaves of the E. indica plant was thought to lack the ability to degrade diuron, resulting in a decrease in mycelial growth and conidial germination.

Herbicide studies on L. theobromae are scarce in the literature. Vazquez et al. (2020) reported that glyphosate had a supstantial impact on soil fungi, especially Diplodia species. The scientists were able to isolate one species of Diplodia from untreated soil, but none from soil treated with glyphosate at twice the recommended dose or at the recommended dose. Similar to glyphosate, imazethapy, topramezone, oxyfluorfen, and diuron significantly reduced L. theobromae conidial germination. Surprisingly, the mycelial development of L. theobromae was either unaffected or minimally influenced by the test herbicides, suggesting that L. theobromae is inferior to B. bicolor due to the high inhibition of conidial germination by test herbicides. In the experiment conducted by Hoagland et al. (2018), it was demonstrated that formulations containing technical grade glyphosate and Myrothecium verrucaria mycelia significantly enhanced the control of Palmer amaranth (Amaranthus palmeri). This result implied that L. theobromae might outperform B. bicolor when their mycelia were used in combination with topramezone, oxyfluorfen, and diuron since no inhibitory effects were observed in these mixtures. Therefore, the combination of L. theobromae's mycelia with these selected herbicides shows promise as a potential mycoherbicide and warrants further testing against E. indica.

5. Conclusions

Environmental factors had minimal impact on the conidia and mycelia of B. bicolor, and its conidia were compatible with topramezone and imazethapyr when used in combination. However, this species exhibited notably slow growth and low conidial germination, which could pose challenges for mass production in mycoherbicide development. On the other hand, L. theobromae is a fast-growing species, although its mycelium showed some sensitivity to temperature and pH fluctuations. Nevertheless, this sensitivity is not expected to hinder future mass production of mycoherbicides. Significantly, L. theobromae's mycelia demonstrated high compatibility with various herbicides, offering flexibility in pathogen-herbicide combinations. Considering these factors, L. theobromae stands out as a suitable candidate for tank mixing with topramezone, imazethapyr, diuron and oxyfluorfen for development of an integrated control of E. indica.

• Funding
  This research received no external funding.

Acknowledgements

The authors are grateful and would like to thank Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA for providing all the facilities to conduct this research, and Universiti Putra Malaysia for partially funding this research with a research grant, vot numbered 6388500-10201.

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Edited by

• Approved by:
  Editor in Chief: Carlos Eduardo Schaedler
  Associate Editor: Michaela Kolářová

Publication Dates

• Publication in this collection
  08 Apr 2024
• Date of issue
  2024

History

• Received
  20 Nov 2023
• Accepted
  11 Feb 2024