Compatibility of Metarhizium sp. (M201-NL strain) in Insecticide and Herbicide Mixtures

Field application of Metarhizium to control Oryctes rhinoceros in oil palm plantations may be overlapped with pesticides (insecticide and herbicide) spray. These chemicals may harm the fungus leading to poor virulence. This paper, however, shows that Metarhizium sp. M201-NL strain may react differently against some pesticides tested using poisoning media method. In the first experiment, the growth of mycelium of the Metarhizium sp. was observed in plated media mixed with insecticide. The highest mycelium radial growth was Fipronil 0.15 % (51.9 mm) which was not significantly different compared to the control, without insecticide (52.2 mm). However, suppression of growth of mycelium was observed in other insecticides. The radial growth of mycelium was significantly lower compared to the control if either Ethiprol, Lambdha cyhalothrin or Acephate was applied at 14 DAI. The second separate experiment involved herbicides. It was found that some herbicides did not inhibit the radial growth of mycelium. These include Metsulfuron-methyl 0.098 % (84.0 mm), Metsulfuron-methyl 0.19 % (82.75 mm), Glyphosate 19.6 % (82.5 mm), Glyphosate 9.8 % (81.6 mm) and Metsulfuron-methyl 0.39 % (80.37 mm) which were not significantly different compared to control, without herbicide (86.12 mm) at 21 DAI. In contrast, the growth of mycelium was significantly retarded when Paraquat, Triclopyr, Glyphosate, and 2.4-D was applied separately. Further study was undertaken to observe sporulation and virulence (bioassay) of the treated mycelium. The sporulations of mycelium treated with Fipronil 0.15 % or Metsulfuron-methyl of different concentrations were not statistically different to control. Similar results were also shown from bioassay. The infection rates of O. rhinoceros larva treated with mixtures of Metarhizium sp. spores with either Fipronil or Metsulfuron-methyl were insignificantly different compared to the control.


Introduction
Growing public concern about the negative impacts of pesticides usages has increased the development of alternative means to control pests in agriculture including the use of Metarhizium sp. as part of integrated pest management (IPM).This entomopathogenic fungus also known as green muscardine is soil borne and rhizosphere colonizing fungus in various ecosystems [1].There are two varieties of Metarhzium anisopliae, i.e. var.anisopliae and var.major.Its morphological characteristics are 5.0-8.0 µm (var.anisopliae ) and 9-15 µm (var.major).The latter is mostly used to control Oryctes rhinoceros (Coleoptera: Scarabaeidae: Dynastinae) [2] which is a serious pest in oil palm and coconut plantations.
There is a potential for large-scale application of Metarhizium sp. to control O. rhinoceros in oil palms.However, various factors are yet to be improved when handling the fungus in the field to get consistently high efficacy.It has been shown that 100 % mortality of O. rhinoceros when treated with 1308 (2024) 012055 IOP Publishing doi:10.1088/1755-1315/1308/1/012055 2 Metarhizium sp.under laboratory conditions, but the efficacy was only 37 or 51 % when it was applied in the field depending on the formulation (dry or wet) [3].Higher efficacy, however, has also been reported that 73 % mortality could be achieved when a dry formulation (powder) of Metarhizium sp. was applied on chipped trunk heaps [4].In addition to differences in its virulence, several environmental factors may cause the great variability of the efficacy of entomopathogenic fungus in the field, including moisture and exposure to chemicals such as pesticides.A previous study on M. rileyi indicated that herbicides Aramo® and Glyphosate® at low and high dosages, respectively, significantly inhibited vegetative growth in vitro [5].It was also shown that conidia sporulation was inhibited by various herbicides i.e.Flex®, Glifosat®, Aramo®, and Basagran.Other studies showed that vegetative growth of M. anisopliae was inhibited by insecticides such as imidacloprid, deltamethrin, cypermethrin, thiodicarb, and chlorpyrifos [6,7] with higher concentrations tend to be more deterrent [7].
Pesticides of different types are commonly used in oil palm plantations to control weeds, pests, and diseases from the start of operation (land preparation) up to immature and mature stages.This practice may hinder the efficacy of entomopathogenic or bioagents applied in the field for various purposes including Metarhizium sp to control O. rhinoceros.However, as discussed above, the response of the fungus may vary depending on the type of strain, chemicals, and dosages used.Our research program at Asian Agri R&D has resulted in the collecting Metarhizium sp.isolates (M201-NL) from Negeri Lama Estate, Bilah Hilir, Labuhan Batu, North Sumatra in 2011.The isolate has been used in the production of ready-to-use formulation and tested in the field to control O. rhinoceros with various success.It was thought that pesticides may harm the fungus and therefore reduce its virulence.The aim of the present study is identifying the inhibitory effects of different herbicides and insecticides on the vegetative growth and sporulation of the M201-NL strain and its efficacy to infect O. rhinoceros.

Metarhizium sp. 201-NL strain collection
Metarhizium sp.isolates used in this study were the M201-NL strain previously collected from Negeri Lama Estate, Bilah Hilir, Labuhan Batu, North Sumatra in 2011.The isolate was screened and propagated using rice media and stored in a referigerator at -5 to 5 ºC after drying.The isolates were refreshed to maintain their virulence by periodically inoculating them to O. rhinoceros larvae every 6 months.

Vegetative Growth Test
Two separate experiments were undertaken to evaluate the effects of herbicides and insecticides on the growth of Metarhizium sp.M201-NL strain using poisoned in vitro media technique.Each experiment was set up using a complete randomized design with 4 replicates.
The prepared media in petri dish were then inoculated with pure of Metarhizium M201-NL mycelial plug with a size 0.5 cm x 0.5 cm.They were then incubated in the dark at room temperature (25-27 ºC).The growth of mycelium was observed by measuring its diameter at 4 different sides every 24 hours until no further growth and sporulation were noticed.Photos in fixed arrangement were also taken for the assessment of mycelia growth.

Bioassay
Further study was undertaken to evaluate the virulence of the Metarhizium M201-NL previously treated with either herbicides or insecticides.Larvae of O. rhinoceros (instar 2) were collected from empty fruit bunch (EFB) heaps in oil palm plantation at Bahilang Estate, Serdang Bedagai, North Sumatra.The larvaes were surface sterilized using sodium hypochlorite solution (0.3 v/v) for 30 seconds, followed by double rinsing with distilled water and drying.They were then placed in test boxes (25 individuals per box) containing oil palm trunk chips and fiber taken from EFB.The chips and fiber were previously sterilized in an autoclave at 121 ºC, 25 Pa for 30 minutes to ensure no pathogens from the field would survive that might hinder the growth of Metarhizium sp.201-NL strain and O. rhinoceros larvae.
The Metarhizium sp.M201-NL strain cultures previously treated with either herbicides or insecticides as explained earlier were propagated using solid rice-based media.Only selected cultures, however, were propagated for this study, i.e. those of showing good adaptation against herbicides or insecticides.The propagated cultures were then used for this bioassay.The concentration of Metarhizium sp.M201-NL strain inoculum in the rice-based media formulation was 10 % w/w.Each test box was then inoculated by evenly sprinkling this formulation of cultures (500 g/box) on the surface of trunk chips and EFB fiber.As a control, O. rhinoceros larvae was inoculated using Metarhizium sp.M201-NL strain formulation from non trated culture.
The boxes were then covered with wire mesh to prevent the introduction of other O. rhinoceros.This bioassay experiment was set up using a complete randomized design with 4 replicates.The observation was made every 24 hours to see the mortality of O. rhinoceros larvae due to infection of the Metarhizium sp.M201-NL strain.

Data Analysis
Data obtained from in vitro tests and bioassay were tabulated using Microsoft Excel 2010 software, then data were processed using IBM SPSS Statistics 26 software for ANOVA.The treatment means were compared by using Duncan's Multiple Range Test (DMRT) at a 0.05 probability level.

Effects of Herbicides and Insecticides on Vegetative Growth of Metarhizium
3.1.1.Herbicides.All herbicides tested in the poisoned media inhibited the vegetative growth of Metarhizium sp.201-NL strain as indicated by a significant reduction of radial growth of the mycelium compared to that of control (Table 1 and Table 2).This inhibitory, however, was only at early stage (1 DAI to 14 DAI).The growth of the inoculum at 15 DAI has recovered in the media containing Metsulfuron methyl (0.098 %) (Table 3).At 19 DAI to 21 DAI, the inoculum showed even higher adaptation to herbicide treatments as indicated by the growing size of radial growth of mycelium in the media containing all concentrations of Metsulfuron Methyl and Glyphosate and particularly at the low concentration (9.8 %).
Other herbicides including Paraquat, 2.4-Dimethyl amine, Flouroxyphyr and Triclopyr at all concentrations and Glyphosate 39.2 % showed permanent inhibitory effects on the growth of Metarhizium sp.M-201 NL strain mycelium.Their radial growth was significantly low compared to that of a control up to 28 DAI.The highest inhibition occurred in the herbicide with the active ingredient Triclopyr at all concentrations, followed by 2,4-Dimethyl amine (at 9.8 % and 19.6 %), Paraquat (1.98 %, and Glyphosate (39.2 %).A previous study has also shown the inhibitory effects of herbicides (Flex®, Gliphosat®, Aramo® & Basagran®) on the vegetative growth of Metarhizium rileyi [8].They also reported significant reduction in conidia production when the herbicides were added into the media except Flex® and Basagran® at low dosages.Other studies on Beauveria bassiana indicated that herbicides with active ingredients of foramsulfuron, tembotrione, and Smetolachlor at concentrations of 50 -100 % had a strong fungistatic effect (> 75 % growth inhibition) [9].The mechanism of inhibition by herbicides is the occurrence of a detoxification system or mycosis in the fungus to limit contact with the herbicide.Mycosis is induced by the active ingredient of the herbicide which in contact with the spores which causes some spores to lack the energy to germinate and even die.Mycosis resulted in only a small number of germinating spores requiring more time to produce colony-sized colonies in controls [10].
Similar results were also seen from the photographs of the cultures (Figure 1).The Metarhizium sp.M201-NL strain showed that herbicide with active ingredient Metsulfuron Methyl did not significantly inhibit sporulation, which could be observed from the dark green color of the colonies of the Metarhizium sp.M201-NL strain (Figure 1.e).In the case of Glyphosate, however, Metarhizium sp.M201-NL strain was only able to adapt to its vegetative but its sporulation was inhibited (Figure 1.b).The Metarhizium sp.M201-NL strain grown on media containing Flouroxyphyr 2.4 %, 4.9 %, and 9.8 % showed better sporulation than those of Paraquat, Glyphosate, 2.4-Dimethyl amine, and Triclopyr at all concentrations.
The ability of Metarhizium sp. to tolerate the inhibition caused by herbicides can be explained as Metarhizium sp.secreting low molecular weight destruxins (dtxs) called secondary metabolites, mainly in response to environmental conditions.Dtxs is a cyclic hexadepsipeptide that can be isolated from Metarhizium sp.More than 40 types of dtxs have been reported, of which dtx A, B, and E are the most significant in the pathogenesis process.Dtx exhibits various biological properties such as antimicrobial, antiviral, antiproliferative, cytotoxic, and immunosuppressive properties which can help the process of infection in the host or media [10].

Insecticide. The test results
showed that all insecticidal active ingredients used in this test inhibited the vegetative growth of Metarhizium sp.M201-NL strain except for Fipronil (Table 4).Insecticides with active ingredient Lambdha Sihalothrin 2 % gave the highest inhibitory effect followed by Achephate-A 0.2%, Achephate-B 0.2 %, Lambdha Sihalothrin 0.25 and Ethiprol 0.15 %.The Metarhizium sp.M201-NL strain appeared to be able to adapt to Fipronil 0.15 % from the start of inoculation up to 14 DAI as indicated by the radial growth of its mycelium which was not significantly different from the Metarhizium sp.M201-NL strain grown on control media.Other insecticides showed consistent inhibition from the start of inoculation up to 14 DAI (Table 4 and Table 5).Although vegetative growth of the inoculum was mostly inhibited by insecticides, the sporulation was still fairly good such as in the media containing Lambdha Sihalothrin 0.25 %, Ethiprol 0.15 %, Achephate-A 0.2 % and Achephate-B 0.2 %.The sporulation was observed in the dark (dense) green spore in the media (Figure 2).However, a higher concentration of Lambdha Sihalothrin (2 %) caused sporulation to fail completely in 14 DAI.This conflicting phenomenon of vegetative growth and sporulation has also been reported that inhibition of vegetative growth did not necessarily decrease sporulation or conidial viability and vice versa [11].The present study has also shown that Metarhizium sp.M201-NL strain tolerated the fungicidal effects of insecticides added into the culture media at lower concentrations but not at higher ones.Similar results have also been reported on M. anisopliae [12].A lower concentration of hexaflumuron and pyriproxyfen (10 %) did not inhibit vegetative growth but higher concentrations (hexaflumuron at 50 ppm and 120 ppm; pyriproxyfen at 500 ppm and 1000 ppm) reduced the growth up to more than 60 % compared to the control.
In nature, like other entomopathogenic fungi, conidia of Metarhizium sp.M201-NL strain may has the same adaptive ability to external environmental stress including chemicals.The present study have It has been shown that the response of Metarhizium sp.M201-NL strain to herbicides and insecticides varied depending on the active ingredients and concentrations.Some pesticides may only inhibit the inoculum growth at an early stage and still underwent sporulation, while others like Paraquat, 2-4 Dimethyl amine exhibited permanent harm (Table 9).Glyphosate at lower concentrations (9.8 and 19.6 %) was temporarily inhibitive but high concentration (39.2 %) caused permanent inhibition.This implies the need to consider the right time to apply either pesticide or Metarhizium sp.M201-NL strain to avoid overlapping that may harm the fungus.Alternatively, the selection of pesticides used may also prevent a negative impact on the selected strain of Metarhizium sp.201-NL strain.Some pesticides may be compatible with the fungus and therefore it can grow and sporulates despite the presence of such chemicals in the field.These options would then give more opportunities to apply Metarhizium sp.M201-NL strain as part of integrated pest management (IPM).

Conclusion
Metarhizium sp.M201-NL strain collected from Negeri Lama Estate, North Sumatra, can tolerate the presence of herbicides and insecticides in poisoned test media depending on the active ingredient and dosage used.Paraquat, Flouroxyphyr, Triclophyr cause permanent inhibition to Metarhizium sp.M201-NL strain.At low concentration, some pesticides like Glyphosate and Lambdha Sihalothrin may only cause inhibition on vegetative growth of Metarhizium sp.M201-NL strain at an early stage, further growth and sporulation can still occur, while at high concentrations exhibit the permanent deterrent effect.Selectivity of Metarhizium sp.M201-NL strain to pesticides under laboratory conditions makes the possibility to apply the fungus to control O. rhinoceros in the presence of such chemicals, but further study is needed to prove the compatibility of Metarhizium sp.M201-NL strain with pesticides under field conditions.
1308 (2024) 012055 IOP Publishing doi:10.1088/1755-1315/1308/1/01205510 All infected O. rhinoceros in the test boxes including control displayed the same typical symptoms of Metarizium sp.infection.It started with the appearance of necrotic spots on the cuticle of the larvae, then the larval body underwent mummification from the inside which could be detected from the hardening of the larval body without any appearance of mycelium on the cuticle (Figure 3.).

Figure 3 .
Figure 3. Infection caused by Metarhizium sp.M201-NL strain in the bioassay test box.

Table 1 .
Mycelium radial growth of M201-NL strain on media treated with herbicide at 1 to 7 day after treatment (DAI).

Table 2 .
Mycelium radial growth of M201-NL strain on media treated with herbicide at 8 to 14 day after treatment (DAI).

Table 3 .
Mycelium radial growth of M201-NL strain on media treated with herbicide at 15 to 21 day after treatment (DAI).

Table 4 .
Radial growth of mycelium M201-NL in culture media containing insecticide at 1 to 7 days after inoculation (DAI)

Table 5 .
Radial growth of mycelium M201-NL in culture media containing insecticide at 8 to 14 days after inoculation (DAI)

Table 9 .
Simplified results of trials on effects of herbicides and insecticides on Metarhizium sp.M201-NL strain in vitro and subsequent efficacy to control O. rhinoceros