Introduction

Eleusine indica L. Gaertn., also known as goosegrass is primarily an agricultural and environmental weed (Randall 2012). It is one of the world's five most problematic weeds, affecting the productivity of 46 different crop species in over 60 countries (Holm et al. 1977). Since it has no root at the nodes, E. indica can be easily removed by hoeing in the early stages of growth. However, when the E. indica matures, a solid root structure forms in the soil, making manual removing difficult. It can withstand a wide range of salt stress, pH and water stress (Ismail et al. 2002, 2003; Chauhan and Johnson 2008). Furthermore, after two years, E. indica seed buried at a depth of 20 cm still had 79% viability (Chuah et al. 2004a).

Glyphosate in combination with fluzifop or sethoxydim (Chuah et al. 2004b), ametryn combined with glufosinate (Chuah et al. 2008a) and tank mixing of monosodium methanearsonate and diuron (Sim et al. 2018) have been effective against E. indica. However, due to antagonistic behaviour, a mixture of glyphosate and glufosinate is not recommended for E. indica (Chuah et al. 2008b). Herbicide resistance in E. indica has evolved as a result of the excessive use of the same herbicides. For example, paraquat, glufosinate, fluazifop-butyl and glyphosate resistance has been demonstrated in E. indica (Dilipkumar et al. 2020). Adopting a diverse integrated weed control strategy that combines chemical, biological, and physical approaches could help to delay the evolution of herbicide resistance (Harker and O’Donovan 2013). Several studies have been done to minimize herbicide use for delaying the evolution of herbicide-resistant E. indica (Chuah and Kent 2021a, 2021b). For instance, the emergence and shoot growth of E. indica were inhibited by 85 to 100% when treated with oil palm frond (OPF) residues at 3 t/ha, implying the possibility of using OPF residues as organic mulches for the weed management programme (Dilipkumar et al. 2015). Further study by Chuah et al. (2018) revealed that the residue of OPF have the potential to reduce the application rate of herbicide S-metolachlor without compromising on the excellent control obtained in combating the herbicide-resistant biotypes of E. indica. However, to date, there is still limited study on a combination of biological and chemcial methods for E. indica control.

Determining the impact of herbicides on fungal pathogens is a key factor in developing integrated weed control strategies (Charudattan 2001). When applied sequentially or as tank mixture, herbicides with synergistic potential cannot be harmful to fungal pathogen spores and mycelium (Hoagland 1996). Herbicides have been thoroughly investigated as methods for combining mycoherbicides to improve the efficacy of weed management. Peng and Wolf (2011) documented that herbicide have been shown to disrupt protective mechanisms of weeds, rendering them more vulnerable to mycoherbicide infections.

Bipolaris setariae, Pyricularia grisea (Figliola et al. 1988), Phoma herbarum and Bipolaris sorokiniana (Maizatul-Suriza et al. 2017) and Bipolaris bicolor have all been isolated from diseased E. indica (Fakri et al. 2019). Pathogenicity tests revealed that Bipolaris sorokiniana infected E. indica on day five after artificial inoculation and had the highest disease severity as compared to P. herbarum and C. aeria after 40 days of treatment (Ismail et al. 2020), none of these fungal pathogens, however, have been evaluated in conjunction with herbicides to combat E. indica. The genus Bipolaris consists of a variety of well-known fungal pathogens that are found all over the world. Leaf spots, leaf blight, dense melting, lateral root rots, and other disease symptoms are common in this species, particularly in crop fields for the Poaceae family, which includes rice, corn, wheat, and sorghum (Berbee et al. 1999).

For all pathogens, there is no universal chemical synergist (Hoagland 1996). There is also a lack of studies on the interactions of herbicides with E. indica-fungal pathogens. Thus, the goals of this study were to 1) assess in vitro compatibility between Bipolaris bicolor and three selected herbicides and 2) evaluate the in vivo efficacy of a combination of B. bicolor and a selected herbicide on inhibition of E. indica seedling.

Materials and Methods

Isolation of putative Bipolaris bicolor

The putative Bipolaris bicolor was isolated from diseased E. indica plants based on the modified method of Maizatul-Suriza et al. (2017). Leaf samples of E. indica infected with leaf spot disease were collected 4 meters apart from each other in corn fields at Research Farm, Bukit Kor campus, University of Malaysia Terengganu. The leaf samples were cut into 0.5 cm × 0.5 cm squares using a sterilized scalpel in a designated leaf region (two-thirds of contaminated area, one-thirds of healthy tissue). For 5 min, the samples were surface sterilized in sodium hypochlorite (NaOCl) (10–20% Chlorox). In sterile conditions, the selected leaf samples were surface-dried on sterilized filter paper. The leaf samples were put in a petri dish with potato dextrose agar (PDA) media, sealed with parafilm and incubated at room temperature for 10 days at 27°C. To obtain the pure culture, actively growing mycelium was sub-cultured onto fresh PDA using a sterilized cork borer or scalpel. After 15 days of incubation, the developing mycelium was looped, then placed on a microscope slide and covered with a cover slip to observe the morphology of the spore under 400× magnification.

Identification of putative Bipolaris bicolor

The total genomic DNA was extracted from overnight culture of fungi isolates using DNeasy Plant Mini kit (Qiagen, Hilden, Germany). Fragment of the gene of interest, was amplified using standard PCR protocol and the universal primers with Thermal Cycler machine. Then, the PCR products was analysed by electrophoresis on a 1% agarose gel, stain with SYBR Safe DNA gel stain. The bands were visualized under Life Technologies E-Gel Imager (Thermo Fisher Scientific, Inc., United States). The PCR products were then further analysed by Apical Scientific Sdn. Bhd. Sequence similarity was estimated by searching the homology in the GenBank DNA database and the National Centre for Biotechnology Information (NCBI) using Basic Local Search Tool (BLAST).

Compatibility of Bipolaris bicolor and selected herbicides

The sub-cultured fungi were flooded with 0.8% Tween 80 solution and scraped on the 15^th day of incubation to carry spore into suspension (Kimaru et al. 2018). To extract the spores, the suspension was filtered through a double layer of muslin fabric. A haemocytometer was then used to determine the spore concentration. The spore suspension at 1.7 x 10⁸ spores/mL was mixed at one-fifteenth recommended rate with flumioxazin, sodium chlorate, or ametryn. This herbicide rate was selected based on our preliminary testing that revealed the chosen rate could provide partial injury to goosegrass seedlings according to the method of Peng and Byer (2005). The mixture was swabbed onto a PDA plate with a sterilized cotton swab and incubated at 27°C for 3 days to see if mycelial growth was inhibited. Mycelial growth was expressed as percent of inhibition. A 0.8% Tween 80 solution with spore only acted as a negative control, while other treatments included 0.8% Tween 80 solution containing spore mixed with flumioxazin, sodium chlorate, or ametryn. The experiment was set up as a five-replicate complete randomized design.

Inhibition of mycelial growth was estimated as follows:

Inhibition area = MG – TMG × 100

Where,

MG – Area of PDA plate covered by mycelium in control

TMG–Area of PDA plate covered by mycelium in treatment

Post emergence application of ametryn in combination with Bipolaris bicolor.

Seeds of glyphosate-resistant biotypes of E. indica were provided by Dr. Cha Thye San (Franci et al. 2020), scariﬁed by using sandpaper and soaked overnight with 0.2% potassium nitrate. The seeds were planted in trays with commercial soil potting mixture (28 cm × 56 cm). E. indica reached the 3 to 4-leaf stage after two weeks. The seedlings were transplanted into a 6 × 9-inch polybag filled with a 3:2:1 mixture of topsoil, cow dung and sand. Using a hand sprayer, E. indica seedlings were sprayed with 2 mL of 0.8% Tween 80 solution, B. bicolor spore suspension at 1.7 × 10⁸ spores/mL, ametryn at one over fifteen recommended rate or a combination of the spore suspension and ametryn. To serve as an adjuvant, 10% edible palm oil was combined with all treatments. The in vivo experiment was arranged as complete randomized block design with five replicates. After spraying leaf greenness was measured using SPAD meter while plant height of E. indica was accessed daily for one week. The leaf greenness was expressed as a SPAD value. One week after treatments, the shoot dry weight of E. indica seedlings was determined. Each plant was carefully removed, washed under running water, and cut to separate the shoot from the root. The above-ground tissues of the plants were then dried for one week at 60°C in a digital oven.

The following formula was used to measure the dry weight reduction of the shoot (Chuah et al. 2008a).

Shoot dry weight reduction =

100-[(treated shoot dry weight/untreated shoot dry weight) × 100)]

Colby's approach is a useful method for evaluating the efficacy of pathogen-herbicide combinations (Peng and Byer 2005). The shoot dry weight reduction of E. indica seedlings was used to assess the association of the natural mycoherbicide, B. bicolor with ametryn. The interaction was considered synergistic when the actual percentage shoot dry weight reduction was at least 5% greater than predicted. The relationship was classified as additive or antagonistic if the change was less than 5% or the actual percentage shoot dry weight reduction was lower than predicted (Grant et al. 1990).

The equation used for calculating the expected response was as follows (Colby 1967):

E = 100 - [(100 - x) × (100 - y)/100)]

Where,

E = Expected response of shoot dry weight reduction as a percentage of control

x = Shoot dry weight reduction as a percentage of control from ametryn treatment

y = Shoot dry weight reduction as a percentage of control from B. bicolor treatment

Mycelia growth inhibition data were analysed using a one-way ANOVA. Tukey's Honestly Significant Difference test was used to compare means at a 5% level of significance. The shoot dry weight reduction data was transformed into an arcsine square root. Data of plant height, leaf greenness, and transformed shoot dry weight reduction data before subjected to a one-way ANOVA, with means compared using Tukey's Honestly Significant Difference test at a significance level of 5%.

Results

Isolation and identification of Bipolaris bicolor

Two strains of fungal pathogens (BR and BF) were successfully isolated from E. indica and identified as Bipolaris bicolor. All samples were blasted from NCBI and showed high similarity ranging from 99.46 to 99.65% from all GenBank descendent throughout world region. Bipolaris bicolor strain CPC 28825 (Accession Number: MF490805.1), which was isolated from the Poaceae family in Thailand, had 99.65% similarity to strain BF. Meanwhile, strain BR was found to be 99.65% similar to B. bicolor strain CPC 28811 (Accession Number: MF490804.1), which was isolated from E. indica in Thailand (Table 1). Thus, the strain BR was chosen to be studied in the following experiments. After 15^th days of growth in PDA, the morphology of mature B. bicolor spore was observed (Fig. 1). The spores of B. bicolor are 1.006 mm long, oblong in shape, and have 2 to 3 septa in the centre at 400× magnification. The spore has septa and is flat, upright, and rarely branched.

Effects of selected herbicides on mycelial growth inhibition of Bipolaris bicolor

Fig. 2 presents the inhibitory effects of flumioxazin, sodium chlorate, and ametryn on B. bicolor mycelial growth in vitro. These herbicides were tested on B. bicolor mycelial growth at a concentration of one-fifth of their respective recommended rates. Flumioxazin was found to be the most phytotoxic herbicide to the B. bicolor fungal pathogen, followed by sodium chlorate and ametryn. Flumioxazin inhibited mycelial growth by 80%, while ametryn inhibited mycelial growth by 10%. This result suggests that ametryn is the most compatible herbicide with B. bicolor for integrated management of E. indica using B. bicolor as a biocontrol agent.

Efficacy of a combination of Bipolaris bicolor and ametryn for inhibition of E. indica seedling

Fig. 3 shows the height of E. indica seedlings after being treated with ametryn, B. bicolor spore suspension, or a combination of B. bicolor spore suspension and ametryn for seven days. Both ametryn and B. bicolor alone, as well as a combination of the two, were phytotoxic to E. indica seedlings, with plant height reaching a plateau on day 3 after treatment and thereafter, implying that treated E. indica seedling’s growth was stunted. Non-treated plants, on the other hand, grew steadily in height from day 3 to day 7. The non-treated plants reached a maximum height of 10.2 cm, while the plants treated with B. bicolor, ametryn, and B. bicolor in combination with ametryn reached a maximum height of 5.2, 5.3 and 5.6 cm, respectively, on day 7 after treatment. The Tukey test also showed that on days 5 and 7, after treatment, there was a substantial difference in height between treated and non-treated plants (P < 0.05). During the 7^th days evaluation period, however, there was no significant difference in height between all treated plants (P > 0.05).

Table 1: Identification of Bipolaris bicolor using 16S rDNA sequencing by depositing the sequences into GeneBank in NCBI

Strain	Accession number	ID from NCBI	Source	Origin	Similarity (%)
BF	MF490805.1	Bipoaris bicolor strain CPC 28825	Poaceae	Thailand	99.65
	KY047110.1	Bipolaris bicolor strain HMC3	Lolium perenne	China	99.64
	KY047109.1	Bipolaris bicolor strain HMC2	Lolium perenne	China	99.64
	KY047105.1	Bipolaris bicolor strain HMC1	Lolium perenne	China	99.64
	MF490804.1	Bipolaris bicolor strain CPC 28811	Eleusine indica	Thailand	99.47
BR	MF490804.1	Bipolaris bicolor strain CPC 28811	Eleusine indica	Thailand	99.65
	MF490805.1	Bipoaris bicolor strain CPC 28825	Poaceae	Thailand	99.47
	KY047110.1	Bipolaris bicolor strain HMC3	Lolium perenne	China	99.46
	KY047109.1	Bipolaris bicolor strain HMC2	Lolium perenne	China	99.46
	KY047105.1	Bipolaris bicolor strain HMC1	Lolium perenne	China	99.46

Fig. 3: Changes in E. indica seedlings height after subjected to ametryn, spore suspension of Bipolaris bicolor and ametryn in combination with spore suspension of B. bicolor throughout 7 days of experimental period. Vertical bars represent standard deviation of mean. *Denotes significant difference between treated and non-treated plants within the same day after analysed by Tukey test at 5% of significance level

Fig. 4 depicts the greenness of E. indica leaves as measured by SPAD after treatments with ametryn, B. bicolor spore suspension, and ametryn in combination with B. bicolor spore suspension over the 7-day duration. Both ametryn and B.

Fig. 1: Conidia (co), Conidiophore (cp) of Bipolaris bicolor grown on a PDA plate at 26 ± 2°C

Fig. 2: Inhibitory effects of flumioxazin, sodium chlorate and ametryn on mycelial growth of Bipolaris bicolor three days after incubation at 26 ± 2°C. Error bars represent the standard deviation of the mean. Mean followed by similar letters have significant difference after analysed by Tukey test at 5% of significant level

bicolor alone, as well as a combination of the two, were found to be phytotoxic to E. indica seedlings. The amount of chlorophyll present in treated plants decreased on day 3^rd and thereafter, with the combined treatment showing a faster decline than the other treatment, indicating that all treatments were able to reduce the green colour of leaf. The SPAD value for non-treated plants, on the other hand, remained constant over the course of the 7^th days assessment period. On days 5 and 7 after treatment, the Tukey test showed a substantial difference (P < 0.05) in SPAD value between treated and non-treated plants. However, the SPAD values of all treated plants did not vary significantly (P > 0.05) over the course of the 7 days of experiment.

Fig. 4: Changes in E. indica SPAD value after exposure to ametryn, Bipolaris bicolor spore suspension, and ametryn in combination with B. bicolor spore suspension over a 7-day period. The standard deviation of the mean is represented by vertical bars. *Denotes significant difference between treated and non-treated plants within the same day after analysed by Tukey test at 5% of significance level

Fig. 5: Shoot dry weight reduction of E. indica seedlings at 7 days after subjected to ametryn, spore suspension of Bipolaris bicolor and ametryn in combination with spore suspension of B. bicolor. Vertical bars represent Standard deviation of mean. *Denotes significant difference between treated and non-treated plants after analysed by Tukey test at 5% of significance level

Fig. 5 shows the reduction in shoot dry weight of E. indica seedlings subjected to various treatments. At 7 days after treatment, the phytotoxic effects of ametryn alone, B. bicolor spore suspension alone, and the combination of B. bicolor spore suspension and ametryn varied significantly (P < 0.05) in reducing shoot dry weight of E. indica seedlings.

Discussion

Bipolaris bicolor has a wide range of distribution including Australia, India. Africa, Brazil, Canada, Cote d'Ivoire, Denmark, New Zealand, Nigeria, Swaziland, Zimbabwe (Farr and Rossman 2013), China (Liang et al. 2019) and Thailand (Marin-Felix et al. 2017). The fungal pathogen is commonly found in host plants from Poaceae such as Zea mays, Eleusine coracana, Pennisetum clandestinum, Oryza sativa, Panicum maximum, Sorghum vulgare, Triticum aestivum, Urochloa panicoides and Zizania aquatica (Farr and Rossman 2013) and Eleusine indica (Marin-Felix et al. 2017). The septation characteristics of B. bicolor exhibited in this work are consistent with those found by Manamgoda et al. (2014) who stated that the fungus produced a slightly cobwebby nearly black growth in pure culture on PDA. However, B. bicolor colonisation was fluffy, cottony, whitish dark grey, and undulated in the present study. This fungal pathogen was first reported as causal agent of leaf spot disease on rubber tree (Liang et al. 2019).

Fakri et al. (2019) reported that diuron is more compatible with 0% inhibition than other herbicides including oxyfluorfen and imazethapyr, which gave 90 and 50% inhibition of mycelial growth, respectively, when tested on B. bicolor. Similarly, in comparison to sodium chlorate and flumioxazin, ametryn inhibited mycelial growth of B. bicolor the least. Both ametryn and diuron inhibit photosystem II in plants, which is a similar mechanism of action. By contrast, flumioxazin had a strong inhibitory effect on the fungal pathogen. Flumioxazin is a photo-dependent peroxidising herbicide that inhibits protoporphyrinogen oxidase, an essential enzyme in chlorophyll biosynthesis, causing photo-toxic porphyrins to accumulate (Shaner 2014). Porphyrins are a common alternative to traditional antibiotics, and they have already been shown to inhibit bacteria, viruses, fungi, and protozoa in vitro (Singh et al. 2016).

On day 7^th after treatment, the non-treated plant had the highest SPAD value of 17.3, while the plants exposed to B. bicolor plus ametryn had the lowest SPAD value of 1.4. The plants treated with B. bicolor alone and those treated with ametryn alone had SPAD values of 5.7 and 2.7, respectively. When E. indica seedlings were treated with a combination of B. bicolor and ametryn on day 7^th, the chlorophyll in the leaves rapidly degraded, thereby leading to plant death. Changes in photosynthesis were a major contributor to both impeded light capture and decreased mesophyll carbon dioxide fixation, as shown by the lower SPAD value. A similar symptom has been identified in the infectious phase of Bipolaris oryzae in rice, which has harmed the physiology of the leaf, primarily due to cell damage at the membrane level (Dallagnol et al. 2011).

The findings of this study on reducing shoot dry weight of E. indica seedlings, agree with those of Ismail et al. (2020), who found that the fungal pathogen Bipolaris sorokiniana inhibited E. indica development 40 days after treatment. They also reported that non-target plants were not affected by B. sorokiniana, but the fungal pathogen consistently infected E. indica. Meanwhile, according to Rusli et al. (2015), Phoma herbarum caused 80% of E. indica mortality 35 days after treatment.

The combination of ametryn and B. bicolor inhibited the most, with 94% inhibition, compared to ametryn alone and B. bicolor alone, which inhibited 75 and 50%, respectively, based on shoot dry weight reduction of E. indica. For ametryn plus B. bicolor treatment, the estimated dry weight reduction value was 88%. This estimated value is 7% lower than the actual value of shoot dry weight reduction, meaning that B. bicolor and ametryn work together synergistically. Ametryn is a non-selective, systemic pre-emergence or post-emergence herbicide that inhibits photosystem II at the chloroplast's thylakoid membrane of plants. By binding to D-1 proteins, the herbicide prevents electron transfer and can damage photosynthetic tissues by disrupting the formation of cell membranes and pigments, resulting in nutrient leakage and cellular dysfunction (Reade and Cobb 2002). In other words, ametryn may have damaged E. indica seedlings by compromising their defence mechanism, exposing them to infection by the fungal pathogen, B. bicolor (Peng and Wolf 2011).

The present findings are consistent with those of Peng and Byer (2005), who used the same multiplicative survival model (Colby 1967) to investigate the synergy of fungal pathogen-herbicide interactions on green foxtail. According to Peng and Byer (2005), propanil and quinclorac interacted synergistically with Pyricularia setariae, resulting in a pattern of increased effectiveness and nearly 100% mortality on green foxtail. Peng and Wolf (2011) looked at a variety of herbicides in conjunction with the fungal pathogen Colletotrichum truncatum and discovered that herbicides including 2,4-D, MCPA, clopyralid, and metribuzin greatly synergized the fungus on scentless chamomile.

Conclusion

It is clearly indicated that ametryn was the most compatible herbicide because it had the lowest inhibitory effect on Bipolaris bicolor. Subsequent greenhouse study revealed that that B. bicolor in combination with ametryn acted synergistically and inhibited the glyphosate-resistant biotype of E. indica seedlings, with the treated plants showing a dramatic and greater reduction in shoot growth as compared to those provided by single application of ametryn or B. bicolor. However, the efficacy of ametryn combined with B. bicolor is possibly affected by growth stage of E. indica and environmental factors, glasshouse findings cannot be specifically extrapolated to field conditions. Further research is needed to determine whether the combination of ametryn and B. bicolor can control mature E. indica in the field.

Acknowledgements

This study was funded by Universiti Putra Malaysia (Vot number: 100-IRMI/GOV 16/6/2 (018/2018).

Author Contributions

ZS, MSAH, and CTS designed the research flow; MAF and NFG performed the research and wrote the manuscript. MAF and CTS edited the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

Data presented in this study will be available on a reasonable request

Ethics Approval

Ethical approval is not applicable in this study.

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