Virulence of the endophytic fungus, Trichoderma asperellum, against the brown planthopper (Nilaparvata lugens Stal)

The brown planthopper (Nilaparvata lugens) is the main pest on rice plants. The pest attack causes rice plants to become dry, brownish, and burnt. Brown planthoppers (BPH) can be controlled in various ways, such as by applying the endophytic fungus, Trichoderma asperellum. The study aimed to determine the virulence of five T. asperellum isolates against brown planthoppers. The study was conducted in a completely randomized design with six treatments and five replications. The treatment used T. asperellum isolates, namely SD327, PC21, A116, SD34, S2D11, and control. The concentration of Trichoderma used was 108 conidia/ml. The ANOVA and 5% LSD were used to analyze the data. The results showed that T. asperellum isolates could infect nymphs and adults of BPH. T. asperellum virulence in nymphs showed that all isolates had the same ability to kill the nymphs (40–48%). The percentage of emerged adults was lower in the PC211 isolate (214%). Applying T. asperellum to adults of BPH caused mortality between 30% - 46%. The highest adult mortality was recorded in the PC211 isolate with the mortality rate of 46% with an LT of 9.224 days. These isolate that was most effective in controlling the BPH population.


Introduction
The brown planthopper (BPH), Nilaparvata lugens Stal (Hemiptera: Delphacidae), is a major pest of rice plants and, until now, has always been a major obstacle for rice production in Indonesia.BPH attacks rice at all growth phases, damaging it by sucking plant cell fluids, reducing chlorophyll and leaf protein content, and reducing the rate of photosynthesis.This pest attack causes rice plants to grow stunted; the leaves turn yellow, wilt, and eventually create burnt appearance on rice plants [1,2].Heavy attacks of BPH can result in crop failure [3].The brown planthopper also damages rice plants by transmitting the grass stunt virus [4].
The brown planthopper can be controlled in various ways.Apart from using resistant rice varieties and synthetic insecticides, use entomopathogenic fungi have good prospects.Many entomopathogenic fungi have been developed as biological control agents for Nilaparvata lugens.A number of fungi are known to infect and control BPH, such as Beauveria bassiana [5,6], Metarizhium anisopliae [7,8], and Lecanicillium lecanii [9,10].The large number of studies on entomopathogenic fungi as pest control agents show that the effectiveness of fungi as biological control agents has good potential to be developed because it can reduce the survival of pests and it is environmentally friendly.
One of the fungi that has been widely developed as a promising biological control agent is the endophytic fungus that lives in plant tissue without causing disease symptoms in plants [11].Endophytic fungi receives nutrition and protection of the host plant so that it can produce or induce plants to produce compounds that play a role in plant defense against pests, disease, and environmental stress.Endophytes play a role in improving plant health in unfavorable environment [12,13].Endophytic fungi are known to be able to change plant characteristics, such as resistance to environmental stress, physiological changes, production of phytohormones, and other chemical compounds [14].Trichoderma is an endophytic fungus that has been developed for pest control.Trichoderma fungi can live endophytically on various types of plants such as rice [15], onions [16], Leucas aspera Arabidopsis, Nicotiana attenuate [18], and mangroves [19].Trichoderma can kill insects through parasitism mechanisms and produces secondary metabolites that are insecticidal as well as antifeedant compounds and repellent metabolites.Trichoderma has the ability to actively parasitize insect hosts and use their bodies as a source of nutrition for the development of new conidia [20].Berini et al. [21] reported that the T. viride fungus produces a chitinolytic enzyme complex that can damage the structure and permeability of the peritropic membrane of the digestive tract of B. mori larvae.As a result, larval growth and development are hampered, pupa weight is reduced, and larval death also occurs.
The use of Trichoderma fungi for controlling insect pests has been reported.Anwar et al. [22] reported that Trichoderma longibrachiatum can kill Bemisia tabaci nymphs.Nymph mortality reached 73% when observed 144 hours after fungus application.Nymph mortality is strongly influenced by conidia concentration.T. longibrachiatum also infect adult stages of B. tabaci.Entomopathogenic activity of T. longibrachiatum was severe on nymphal stage as compared to that of adult B. tabaci.Nasution et al. [23] reported that the Trichoderma fungus caused 100% death in Oryctes rhinoceros, 15 days after Trichoderma application.Application of Trichoderma in the field can cause up to 50% death of Leucinodes orbonalis in eggplant and, in effect, it increase yields by 56%.To support the successful use of Trichoderma to control BPH biologically, the selection of isolates with high virulence is required.The aim of this research was to study the virulence of various T. asperellum isolates against BPH.

Preparation of Trichoderma asperellum
The isolates of T. asperellum used in this study were collected from the Laboratory of Biological Control, Department of Pests and Plant Diseases, Agriculture Faculty, Universitas Andalas.There were five isolates of T. asperellum used in this research.All isolates were isolated from various plants (endophytes) and grown on Sabauraud dextrose agar (SDAY) medium (Table 1).

Host plants and rearing of N. lugens
The rice seed used for rearing of BPH was the IR42 variety from Batu Busuk Village, Pauh District, Padang City, West Sumatra.The rice seeds used for rearing BPH were 50 g/jar.The jars used were 8 pieces with a diameter of 27.5 cm and a height of 27 cm.The seeds to be used are soaked in water for 24 hours and then air-dried for 60 minutes.Furthermore, the seeds are sown evenly into jars and soaked again using plain water.The availability of water is always maintained in a position to cover the rice grains.After 5-7 days, rice seedlings can be infested and used as BPH host plants.
A total of 20 pairs of adults BPH were collected from rice fields in Kuranji sub-district, Padang city using an aspirator and put into plastic cups containing rice seeds for temporary placement of adults BPH.The adults obtained was taken to the laboratory and transferred to a jar containing rice seeds.In order for the obtained BPH stadia to be uniform, approximately 3 days after infestation, all adults were removed from plastic jars, while rice seedlings were maintained until the eggs hatched into nymphs and subsequently became adults.The BPH used was the 8 th generation.The total number of nymphs and adults used in each test was 300 individuals.

Preparation of conidial suspension
Isolates of T. asperellum propagated on SDAY medium in petri dishes at 25 o C for 7 days.Fungal conidia were harvested by adding 10 ml of sterile distilled water and 0.05% Tween 80 as grading material into the petri dish.Conidia were removed from the medium with a fine brush.The suspension was filtered and the conidia concentration was calculated using a haemocytometer.The conidia concentration used was 10 8 conidia/ml.

Virulence test
Virulence test of nymph and adult mortality were carried out separately.A total of 10 nymphs instar II and 10 BPH adults were infested into plastic cups (top diameter = 9 cm, base = 5.5 cm, t = 10 cm) containing a 1:1 mixture of soil media and manure in which five varieties of rice seeds had been planted.IR 42.Application of T. asperellum suspension to BPH is carried out by spraying until the nymphs and adults of BPH are completely wet.Observations were made every day until 7 days after application.Nymphs that successfully become adults are counted to obtain the percentage emerged adults fof BPH.The dead BPHs were re-incubated to prove that the death of the BPH nymphs was caused by T. asperelum by placing the dead BPHs in a petri dish lined with filter paper moistened with sterile distilled water.

Data analysis
The data obtained was analyzed using analysis of variance (ANOVA) and LSD using STAT 8. Lethal period of LT50 T. asperellum against test insects was calculated using SPSS software

Nymph mortality
The results of the virulence test of the four T. asperellum isolates on the mortality of the second instar of BPH nymphs showed that all T. asperellum isolates were able to cause mortality on BPH nymphs.All isolates were effective in causing mortality in the second instar nymphs of BPH (40-48%).Different sources of T. asperellum isolates did not significantly affect nymphs mortality (P<0.0000).LT50 value of T. asperellum range from 8.26-11.61days.PC211 isolates had an LT50 value of 8.26 days, shorter than those of other isolates (Table 2).2, it shows that isolates of did not have a significant effect on mortality of BPH nymph, but they are significantly different from the control.Nymph mortality is still low, only 40-48%.Ahmed and El-Katatny [24] reported that T. harzianum caused up to 80% larval mortality against the Egyptian cotton leafworm Spodoptera littoralis.In contrast, Praprotnik et al (2021) [24] reported that the Trichoderma isolates tested showed little or no pathogenicity against Tenebrio.molitor.
Daily mortality of BPH nymphs increased until the 7 days after application.Mortality of BPH nymphs applied to second instar BPH nymphs occurs at 1-3 days after application.SD327 isolate killed BPH nymphs 1 day after Trichoderma application.Overall, Trichoderma isolates killed nymphs on 2 days after application (dap).Nymph mortality continued to increase until 7 Days after application Figure 1.Daily mortality of instar II BPH nymphs after T. asperellum application from 1-7 days after application

Percentage of emerged adults of BPH
Application of the fungus T. asperellum can reduce the percentage of emerged adults of BPH.The lowest percentage of emerged adults was found in the treatment of PC21 isolate followed by A116 isolate.The percentage of emerged adults by spraying treatment with T. asperellum was lower, namely 44% of PC21 isolates.Emergence of BPH treated with SD11, SD327, SD324, and A116 isolates were not significantly.All isolates, however, produced lower BPH emergence percentages compared to the control (Tabel 3).Means followed by the same letter within the same column are not significantly different at P = 0.05 according to LSD Test

Adult mortality
We showed that all the T. asperellum isolates can infect the adult of BPH.Mortality of BPH adults after application of T. asperellum varies depending on the isolates.PC21 isolate was more effective in increasing adult BPH mortality percentage of 46.00% with LT50 9.224 days.The percentage of mortality in adults is lower compared to nymph mortality.Mortality of adult BPH is lower due to morphological factors of adult BPH.BPH adult stages have a harder cuticle compared to BPH nymphs (Table 4).Table 4. Adult mortality of BPH after application of T. asperellum 7 days after application Adult daily mortality of BPH continued to increase until 7days after application.The mortality occurred one day after T. asperellum application, then continued to increase until 7 dap.On 7 days after application, PC21 isolate had high virulence compared with other isolates (Figure 2).multiplying in the host.Entomopathogenic fungi infect insects through direct penetration of the cuticle, requiring adhesins and lytic enzymes (chitinases, proteases and lipases).Next, the fungus overcomes the insect's immune system and colonizes its body, eventually forming and spreading new conidia from its dead host.Throughout the process, entomopathogenic fungi must produce a variety of insecticidal secondary metabolites that enable the fungus to complete its life cycle [26,27].Kottb et al. [28] reported that the mechanism of the T. asperellum fungus produces a volatile compound, namely 6-pentyl-αpyrone (6PP), with a rate of 450 ng/μ.The 6-pentyl-α-pyrone compound can kill Tetranychus urticae up to 100%, 48 hours after application.Trichoderma viride Pers.Derived chitinases have effectively degraded the chitinous vital structures of Bombyx mori (Linnaeus, 1758) larvae [29].

Isolates
Trichoderma longibrachiatum isolated from cotton mealybug shows entomopathogenic activity against Bemicia tabaci similarly like other species of Trichoderma that have antagonistic activity against different bacteria [30] and fungal pathogens [31,32] .T. longibrachiatum has been previously reported from Cowpea aphid, Aphis Koch [33] and its soil isolate showed entomopathogenic activity against Leucinodes orbonalis which is one of the major pests of brinjal (eggplant, Solanum melongena) [34] .Trichoderma acts directly as an entomopathogen through parasitism mechanism and the production of insecticidal secondary metabolites, antifeedant compounds and repellent metabolites [19].The oral application of T. koningi spores to grass moth larvae (Ostrinia furnacalis) caused the mortality of 30% of larvae in 12 days [34,35].Inoculation of hazelnut branches with spores of T. harzianum, T. asperellum and T. atroviride has been shown to drastically reduce the beetle population [36].

Conclusion
The results showed that the endophytic fungus T. asperellum can infect BPH nymphs and adults.Nymph and adult mortality were influenced by isolates.Apart from being able to kill nymphs, application of the T. asperellum fungus can also inhibit adult formation of BPH significantly.Trichoderma asperellum PC21 is an isolate that is more virulent against BPH.

Figure 2 .
Figure 2. Daily mortality adults of BPH after T. asperellum application from 1-7 days after application

Figure 3 .
Figure 3. (a) Nymphs not infected with T. asperellun (b) nymphs infected with T. asperellum (c) adults not infected with T. asperellum (d) adults infected with T. asperellum Differences in BPH nymph mortality after application of T. asperellum are caused by differences in the physiological, geographic and genetic characteristics of the isolate source used [33] .This research shows that the mortality of BPH nymphs and adults is greatly influenced by the isolate source used.PC21 isolate has better ability and virulence in controlling BPH.Isolate PC21 is an isolate originating from the stem of the Cisokan variety of rice plant which is the host of BPH.Trichoderma acts directly as an entomopathogen through parasitism mechanism and the production of insecticidal secondary metabolites, antifeedant compounds and repellent metabolites[19].The oral application of T. koningi spores to grass moth larvae (Ostrinia furnacalis) caused the mortality of 30% of larvae in 12 days[34,35].Inoculation of hazelnut branches with spores of T. harzianum, T. asperellum and T. atroviride has been shown to drastically reduce the beetle population[36].

Table 1 .
Isolates T. asperellum used in the study

Table 2 .
Mortality of instar II BPH nymphs after application of Trichoderma asperellum 7 days after application UN16.19/PT.01.03/PDKNPangan/2023) of Directorate General of Higher Education, Research and Technology, Ministry of Education, Culture, Research and Technology, Indonesia.