Pesticides and Chemical Fertilizer are not Negatively Impact the Diversity of Entomopathogenic Fungi on Rice Plant in Malang Indonesia

The conventional system of rice plantation has been applied in Malang, Indonesia. Standard chemical fertilizer and pesticide application were used in this field. Soil samples in rhizosphere of rice plantation showed the existence of several entomopathogenic fungi including Penicillium sp, Aspergillus sp, Trichoderma sp and some unidentified fungi. The diversity value also demonstrated a medium diversity. We conclude that an application of pesticides and chemical fertilizer according to recommended practices, are not negatively affect the diversity of entomopathogenic fungi. Aspergillus sp and Penicilium sp can cause death against Spodoptera litura .

environmental condition as well.Various ways of applied cultivation aim to increase the production of rice crops.The use of fertilizers and pesticides cannot be separated from farmers' choice to increase their production and reduce pest populations.Pesticide used should be adjusted with the dose recommendation since an excessive use of pesticides can cause toxicity, lead to pest-resistance to pesticides and pesticide residue in agricultural sites including soil and irrigation water [3]; [4].The environmental pollution caused by continues agricultural activity that rely on chemical fertilizers and synthetic pesticides.The application of conventional system can be found in several regions of Indonesia [5].
Soil is the most important habitat for entomopathogenic fungi, which can contribute significantly to the population control of insects in the ecosystem [6].Microbial community decompose complex organic matters in the soil which then convert them as nutrients to be utilized by plants [7], thus the existence of microbial communities in soil play important role in nutrients availability.Other than that, soil and rhizosphere also function as habitat for various natural enemies of pests including entomopathogenic fungi which are widespread in agro-ecosystems of temperate and semi natural habitats [8]; [7]; [9].Entomopathogenic fungi have been viewed as excellent biocontrol agents Pesticides and Chemical Fertilizer are not Negatively Impact (Handoko, et al.) in the regulation of phytophagous insect and mite populations in agroecosystems [10].The pest control efficacy of various entomopathogenic fungi species has been reported in Hypocreales (Ascomycota), but not limited to Beauveria [11].
The process of decomposition increased the abundance of entomopathogenic fungi, which are negatively impact the insect population such as Aphids on the above ground and soil fauna such as microfauna, bacterivorous, fungi plant feeding nematodes on the under ground [12].Several species of fungi found in both under and above ground are potentially functioning as entomopathogenic fungi [9]; [13].Several entomopathogenic fungi stimulate plant growth and inhibit bacterial pathogens in soil [8].However, information regarding beneficial fungi in rhizosphere of rice plantation, especially entomopathogenic fungi are poorly understood.
In this paper, the diversity of entomopathogenic fungi in rhizosphere of rice plants in the conventional system in Malang, Indonesia were studied.Identification of entomopathogenic fungi as well as their virulence are also discussed.

MATERIAL AND METHOD Study Area
The data collection was conducted in Malang, Indonesia.The rice cultivation practice were conducted according to standard recommendation application as described in Table 1.

Application of Pesticides
The systemic pesticides used in rice cultivation consists of 3% carbofuran.Application of pesticides used was according to those recommended by ministry of agriculture in Indonesia (Table 1).Carbofuran also has contact activity against pests.It is one of the most toxic pesticides still in use.It is marketed under the trade names Furadan.Carbofuran 3G applied after 1 month old rice [14].

Soil Sampling
In order to identify the diversity level of entomopathogenic fungi in rice rhizosphere, soil samples were collected at 3 months old rice in near the roots of rice plants using a trowel.Soil samples were taken at 5 points (25 gram soil) with diagonal system [15].Rhizosphere soil section was taken with a depth of 30 cm [16].Soil samples are a composite from 5 points of experiment area.The diversity index is calculated by the formula Shannon Wienner index [17]:

Isolation of Fungi
Isolation of the fungus was carried out by the dilution plate method [7].For each land location, 25 g of soil samples were taken and dissolved with 225 ml of peptone and then in fortex, a dilution of 10 -1 was obtained.Suspension of 10 -1 dilution is taken 1 ml and dissolved with 9 ml of peptone then in fortex then obtained dilution 10-2.The dilution continues until the dilution level is 10-5.Before culturing in the Sabouraud Dextrose Agar Yeast Extract (SDAY) media, each dilution was carried out fortex to dissolve.Each level of dilution was taken 0.1 ml then cultured in SDAY media in petri dishes and then leveled.The dilution rate ranges from 10 -5 to 10 -1 , as it reduces the deposition of the tip in the micropipette.Then it was incubated for 3 days in an incubator at 35°C.The dilution activity was carried out in a Laminar Air Flow Cabinet (LAFC).After incubation, observations were made on each plate.If there is more than one fungal colony growing on one petri dish, then purification is carried out.Purification aims to separate each macroscopically different colony.The mushrooms were cultured again on SDAY media in tubes and incubated for 3 days in an incubator at 35°C.After the SDAY media in the tube was pure, then it was purified into SDAY media in a cup.Then it was incubated for 7 days in an incubator at 35°C.Purification activities are carried out in LAFC.

Observation Parameter Macroscopic and Microscopic Observations
The purified and prepared isolates of entomopathogenic fungi were then observed macroscopically and microscopically and identified with the guidelines for fungal identification, namely [16].Macroscopic observations were carried out on the 7th day after inoculation by observing the morphology High diversity, the spread of the number of individuals per species is high of the fungal colonies which included colony color, colony surface texture (velvet, powder, cotton and cotton, wrinkled), colony distribution pattern in petri dishes (concentric and not concentric), colony diameter (cm).
Microscopic observations were carried out by observing the morphology of fungal colonies using a microscope which included hyphae shape, conidia shape, conidia distribution pattern, description of fungal parts.Observations were made using 1000x magnification.Observation of the presence or absence of septa on hyphae is done by observing the presence or absence of a septum (cross line).The septa on the hyphae can be seen tightly or rarely.Conidia can be round, oval, elliptical, oval or irregular in shape.Conidia distribution patterns such as clustered at the end of the conidiophores or clustered around the hyphae, spread, single, chained or not chained, as well as the form of conidia collections.Conidia collections are often seen in various shapes, such as round, radial (irregular), resembling the shape of a flower, and etc. Observations of conidiophores include the presence or absence of septa on conidiophores (insulated or not insulated), and the growth of conidiophores (branched or unbranched, long or short).

Genera of Entomopathogenic Fungi Present in Rice Rhizosphere
Based on fungi identification in soil collected from rhizosphere around rice plants in Malang, eleven fungal isolates group were observed.Eleven groups of fungal isolates were identified and resulting three different genera of fungi including Penicillium sp., Aspergillus sp., and Trichoderma sp were collected.Macroscopic and microscopic morphological characteristics were analysed as presented in Figure 1-4 and table 2.

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Aspergillus sp.(isolates b, c, e, f, h, k) Isolates b, c, e, f, h and k macroscopically showed yellowish to dark brown with a white edge (Figure 2a).Meanwhile microscopically showed conida (round-egg), fialid, Metula, vesicles, conidiophores, and hyphae (not insulated) (Figure 2b).The characteristics of this fungus colony also showed in table 2. For the colony Aspergillus sp. 1, its top-colored was black white edge and colonies reserve gray white edges, characteristic of surface colonies were powder ledges wrinkled.The colonies could grow to reach 9 cm and have oval conidia.The conidiophores were fialid and metula.The branching type was monoverticillate and hypha was not insulated.Meanwhile in Aspergillus sp.2, the top-colored was white, black powder and colonies reserve white, For Aspergillus sp. 3, the top-colored was dark brownish and colonies reserve dark brown whitish gray edges, characteristic of surface colonies were powder.The colonies could grow to reach 7,2 cm.For Aspergillus sp. 4 top-colored was white green edge and colonies reserve yellow dark green edged, light brown, and characteristic of surface colonies were powder.The colonies could grow to reach 2,7 cm.For Aspergillus sp. 5, the topcolored was brownish white and colonies reserve white and characteristic of surface colonies were wrinkled velvet edges.For Aspergillus sp.6, the top-colored was white edge yellow and colonies reserve white edge yellow and characteristic of surface colonies were cotton edge powder.These colonies could grow to reach 7,6 cm.characteristics of conidia, conidiofor, branching, fialid, metula and hypha for Aspergillus sp 2, 3, 4, 5, and 6 showed similar to those on Aspergillus sp 1. Morphological identification of Aspergillus species from corn grain in Penang Island, Peninsular Malaysia has characteristics black colony, biseriate conidial heads and small conidia (2,9 -3,9 µm) [7].

 Penicillium sp. (isolates a, d, g, l)
Macroscopic result of isolates a, d, g, l showed that those isolates were included in genus Penicillium sp since the conidia were green and white edge (Figure 1a).This result was similar with the previous research that colony of Penicillium sp. has a green color, sometimes white, mostly have conidiophores.
[11] also demonstrated that the Penicillium sp.found in land of Himalayas, India has the characteristic green color of top and bottom the white and the type of uniform spherical distribution.
In microscopic result, those isolates had konida (round-egg), fialid, conidiophores, hyphae (sectional) and metula (Figure 1b).A detail characteristics showed in table 2. For Penicillium sp. 1, its top-colored was old green edge white and colonies reserve white yellow, and surface colonies were velvet edges wrinkled.Diameter of Penicillium sp. 1 was 3,2 cm and had oval conidia.The colonies had conidiofor, fialid and metula.The branching type was monoverticillate and hypha was insulated.For Penicillium sp. 2, its topcolored was old green edge white and colonies reserve white yellow, and surface colony was velvet edges wrinkled.Diameter of Penicillium sp. 2 was 1,9 cm.For Penicillium sp. 3, its topcolored was green edge white and colonies reserve green edge white, and surface colonies were velvet.Diameter of Penicillium sp. 3 was 6,45 cm.For Penicillium sp. 4, its top-colored was green edge white and colonies reserve white yellow, the surface colony was velvet.Diameter of Penicillium sp. 4 was 1,15 cm.The characteristic of shape of conidia, conidiofor, branching, fialid, metula and hypha for Penicillium sp 2, 3, and 4 were similar to those on Penicillium sp 1.
According to [18], the characteristics of Penicillium sp.including red white color, with brush-like shape, having mycelium with septate shape and fruiting body shape is conidia in chain.
According to [19], that single conidiophores (mononematus) or compound (synematous), consisting of a single rod share some phialid (simple / monoverticillata).All cells among Metula and potentially stem become a branch.Branching one level (biverticillata-symmetric), branching two levels (biverticillata asymmetric / terverticillata), three kinds or more tiers of branches (quaterverticillata). Phialid is a structure that sustains conidia, cylindrical basal section that narrows at neck, or lancoelate (more or less most part embedded in the tip of basal shoots).Conidia form long chains, divergent or column, globular, elliptical or fusiform, transparent or greenish, with walls smooth or bumpy.Thus, based on the result, it was confirmed that a, d, g and l isolates are Penicillium sp.

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Trichoderma sp.(isolates i) A Trichoderma sp. was confirmed based on observation on isolate i.This isolate had yellow and green edges of yellow and white edge of conidia (Figure 3a).Trichoderma spp.from New Delhi has colour from whitish to greenish (either dark or light) during the growth period.After 7 days, culture plates were observed to be dark green in colour and all the isolates [20].Microscopic data showed that genus Trichoderma has conida (round-elliptical), fialid, conidiophore, hyphae (not insulated) (Figure 3b).A more detail characteristic of this fungus was presented in table 2. In Trichoderma sp. 1, its top-colored was yellow green edge, yellow white.The colour of colonies reserve were gray white edges, characteristic of surface colonies were cotton edge powder.These colonies could grow to reach 9 cm and had ellipse conidia.The colonies had conidiofor, fialid and metula.The conidia were branched and the hyphae were not insulated.
According [21] stated that Trichoderma spp. on organic potato plant rhizosphere Indonesia has a smooth-walled conidia, first a colony of hyaline, then becoming greenish white, and then dark green, especially on the show there are many conidia.
Pesticides and Chemical Fertilizer are not Negatively Impact (Handoko, et al.) Conidiophores can be branched resembles a pyramid that is at bottom lateral branches are repeated, while getting to the end of branching are getting shorter.Phialid look slim and long, especially at the apex of the branches.conidia round to oval shaped a short spring. Unidentified (isolates j) Isolates j is group of fungi that have not been identified because it only had conidiophore and conidia (Figure 4b) and is macroscopically white velvet and wrinkled (Figure 4a).Detailed information regarding this fungus showed in table 3.For the colonies of unidentified fungi, its top-coloured and its colonies reserve were white, characteristic of surface colony was velvet edges wrinkled.This colony could grow to reach 8,25 cm and had ellipse conidia.The conidia were branching and had no fialid, metula and hyphae.It was possible that this isolate was included in sterilia mycelia.According to [22], unidentified fungi have mycelium characteristics such as cotton, white or yellow or black, grow fast and some are slow.However further identification is needed.
A B  Pesticides and Chemical Fertilizer are not Negatively Impact (Handoko, et al.)The Entomopathogenic Fungi Shows Medium Diversity In order to find out the diversity status of entomopathogenic fungi based on dilution plate analysis in rhizosphere area in rice paddy plantation using conventional system, calculation of fungal diversity index was conducted.The result showed a medium diversity values (1.13) as shown in Table 4.The calculation of evenness index showed a high category on Aspergillus sp and Penicillium sp which demonstrated value of 5.17 and 3.79, respectively.Whereas a low evenness index categories showed by Trichoderma sp. and unidentified isolates (0.00).A medium diversity index and high evenness index on Aspergillus sp and Penicillium sp might be is most dominant fungi in rice field.Aflatoxins are carcinogenic metabolites produced by several strains of Aspergillus sp and Penicillium sp group in food and feed.Aflatoxin contamination is one of the main factors affecting rice seed quality [20].The medium index here still needs further testing by comparing other organic or conventional soils, the figures obtained from Aspergillus and Penicillium are based on Shannon Wiener's analysis.
Thus, no excessive usage of both chemicals applied in rice cultivation site.Similar to previous research, an application of nitrogen fertilizer on field, when applied according to recommended practices, are unlikely to negatively impact survival of B. bassiana in pecan orchards when the fungus is applied for Curculio caryae suppression during weevil emergence [21].Urea had no significant affect on the fungus.Therefore, it seems that certain factors in manure that reduce survival of Beauveria bassiana are lost during decomposition [23].
However, a very low evenness index in Trichoderma sp. and unidentified isolates might be caused by there was no organic fertilizers such as compost or other organic manure which simultaneously applied in the rice field at all for several years.According to previous research in rhizosphere of Chrysantemum and other temperate crops showed that application of liquid organic fertilizers and manure compost can help to enhance the microbial diversity [22]; [23]; [24].Evenness index values are influenced by the number of species present in the community.A type which has a high degree of stability has a greater opportunity to maintain the sustainability of its kind.To assess the stability of species in a community may use equity index value (E).The higher the E value, then more stable the diversity of species in the community, on the contrary, the lower the E value, then the less stable the diversity of species in the community.Based on this result we may suggest that in order to enhance entomopathogenic fungi diversity in rhizosphere of rice plant, application of organic compound such as organic fertilizers and manure composts are necessary.

CONCLUSION
The diversity index value of entomopathogenic fungi was classified as medium category.It demonstrated that an application of pesticides and chemical fertilizer according to recommended practices, are not negatively impact the diversity of entomopathogenic fungi.However, application of organic fertilizers as well as manure compost is suggested thus it can improve the entomopathogenic diversity.Identified fungi found in rhizosphere of rice plant were Penicillium sp, Aspergillus sp, Trichoderma sp and some unidentified fungi.However, Aspergillus sp had the highest virulence among others.Soil acidity and the low soil fertility are barely impact the diversity of entomopathogenic fungi.
the number of individuals of one species with the number of individuals of the entire sample in the plot (n/N)

Table 1
Land use and rice plantation practice Medium diversity, the distribution of the number of individuals of each species is moderate >3

Table 4
Value of index diversity (H ') and evenness index (E) fungi from rhizosphere of rice plants with conventional systems