Effects of entomopathogenic Aspergillus flavus on tomato plant (Solanum lycopersicum) endophytic activity under agro-climatic condition of Lahore, Punjab-Pakistan

Entomopathogenic fungi can Control population of various insects. A number of entomopathogenic fungi also have ability to endophytically colonize many plant species. The Endophytic colonization by these fungi providesa source of indirect interactions between fungi and insects.This endophytic activity of entomopathogens may contribute in regulation of insect populations but also can cause diseases and damage to the plants. The entomopathogenic Aspergillus flavusis able to colonize different plant species by producing aflatoxins which is more favored in warm temperatures (32-38oC). The present study is aimed to assess the effects of Aspergillus flavuson tomato plant (Solanum lysopersicum) and to determine its endophytic activity. Aspergillus flavuswas isolated from soil using insect bait method. The fungal suspension was prepared and applied on tomato plant leaves by spraying. After 7 days significant disease symptoms related to gray mold disease, late blight and leaf mold were observed when compared to plants of untreated control group. Microscopic observations showed 100% colonization of Aspergillus flavus on treated leaves. It is evident that the Aspergillus flavus can endophytically colonize tomato plant leaves and cause various diseases. So the utilization of entomopathogenic Aspergillus flavus to control insect population needs further research investigation for its environmental safety to non-target organisms.


Introduction
The number of entomopathogenic fungi forms a total of approximately 750 species from 85 genera that are found in all classes of fungi. In almost every order of insects and taxa of arthropods these pathogens can cause mycoses. These fungi are commonly found in terrestrial habitats. However, there are minimal effects of entomopathogenic fungi on non-target organisms and these fungi provide safer alternative to chemical insecticides [1]. Entomopathogenic fungi differ from other microorganisms in causing insect diseases because they infect the insects by penetration through their cuticle, insects do not need to ingest them and have a great potential for controlling the sucking insects such as aphids [2]. The mechanism of pathogenesis of these fungi starts particularly in the areas of formation of an infection structure, entry into the host and toxin-mediated host death [3]. Aspergillus flavus have worldwide distribution and can infest a wide range of agricultural products. Aspergillus flavus produces aflatoxins. The growth of Aspergillus flavus and synthesis of aflatoxins depends on substrate, pH, temperature, moisture, aeration and competing micro-flora [4]. Aspergillus flavus produces airborne conidia, which easily disperse by air movement or by insects. Aspergillus flavus grows better with water activity and the optimum temperature for the growth of Aspergillus flavusranges from 12⁰C to 48⁰C [5]. Although most of the entomopathogenic fungi have no adverse effects on non-target organisms but some strains of Aspergillus flavusare found to be an unspecialized saprophyte along with its pathogenicpotential against various insects [6,7]. Aspergillus species are considered as opportunistic pathogens that take advantage of an opportunity not normally available, such as a host with a weakened immune system, an altered micro-biota for colonization [8]. Fungal entomopathogens are capable of naturally controlling the populations of various insects. A wide range of insects pests in the agricultural crops are efficiently biologically controlled by entomopathogenic fungi [9]. Entomopathogenic fungi infect insect in all stages of life cycle from egg to adult [10,11].The production of secondary metabolites variesamong the different strains of same species of entomopathogenic fungusand affect differently against various target and nontarget organisms [12].Aspergillus flavus has been reported entomopathogenic but due to its non-specificity of host it can also affect various non-target organisms [13]. The main objective of the study was to evaluate the safety of entomopathogenic Aspergillus flavusisolated by employing insect bait method, against tomato plant (Solanum lycopersicum) to explore its potential as safe alternative to chemical insecticides.

Materials and methods Plant material
The experiment was conducted at Lahore College for Women University (LCWU), Lahore, Pakistan after approval of university Board of Studies. The plantswere acquired by local nursery in Lahore, Pakistan to assess the safety of Aspergillus flavus against tomato plant.

Isolation of entomopathogenic fungi from soil
The collection of soil samples was done from Jallo Park, Lahore from 10 inches depth. For the isolation of Aspergillus flavus the insect bait method was used [14]. Baiting of soil sample was done using Culexpipiensto isolate indigenous species of entomopathogenic fungi. Aspergillus flavusspecie was cultivated using Potato Dextrose Agar (PDA) which ensures its mycelial growth and maintains the other growth factors. The Aspergillus flavus colonies were fully grown after 5-6 days.All procedures were carried out at room temperaturei.e. 25 to 30⁰C.Aspergillus species grown on PDA media were observed (with naked eye) for identification of morphological characteristics. Initially the colony growth was low and mycelium acquired white color. The mycelium acquired green to olive green color after 3-5 days of incubation and also the colony growth was increased. The colony was plain flat at the edges but elevatedin the centers [15]. Scanning electron microscopy of conidia was also performed (Fig. 1). The conidial heads observed under microscope typically radiate longitudinally, conidiosporesare uncolored, coarsely roughened, approximately less than 1mm long. The samples were further identified for macroscopic and microscopic morphological characteristics by the experts.The Aspergillusflavus conidia are hydrophobic in nature. To prepare a conidial suspension, 0.01% tween80 solution was added to ensure its uniform mixing in water. The conidial suspension of Aspergillus flavus was prepared to count the no. of conidia per ml on hemocytometer (MARIENFELD SUPE-RIOR, Germany) using the microscope (NIKON DS-L2, Japan) and for further dilutions needed for the experimentation.The conidia of Aspergillus flavus were counted on the 25grid chamber. Three dilutions of 10 5 , 10 6 and 10 7 were prepared from the conidial suspensions as per the requirement of experiment. Application of Aspergillus flavussuspension on tomato plant 24 tomato plants were obtained out of which 18 plants were allocated to experimental group. Three experimental groups with 6 plants in each group and 6 plants as the control group were used for the study. External contamination and fungal suspension runoff was prevented by covering the base of plant by aluminum foil and plastic cover. Four treatments were used in the test, 0.01% tween80 solution for control group and three different concentration of conidial suspension 10 5 10 6 and 10 7 . 3ml from each suspension solution was taken in the sprayer and applied onto the leaves of plants one by one. The humidity of plants was maintained.After 7 days observation the leaves were randomly selected from each plant.Each leaf from experiment group and control group plant was disinfected. Six leaves per treatment were disinfected by first washing them with running tap water and then submersing them in 70% ethanol for 15 sec, followed by 20 min in 5% sodium hypochlorite and rinsing 10 times in sterile distilled water.Leaves were cut to form small leaf sections (3 sections of each leave) of 1 x 1 cm. Each leaf section was placed on PDA plate and kept in fume hood. The data was expressed as colonization frequencies.
Colonization frequency = 100 x No. of plant sections colonized/ Total No. of plant sections The morphological characteristics of conidia grown on treated leaves were microscopically examined by experts to identify the incidence of Aspergillus flavus grown on treated tomato plant leaves.

Results
After 1 day of exposure no significant changes were observed in plants.
Aspergillus flavus did not cause any physical change in the plant. After 4 days of exposure slight changes were observed in plants. Leaf curling and formation of small greenish-yellow spots near the margins of leaves were observed in 10 7 treatment plant leaves which showed that Aspergillus flavus has started its endophytic activity. After seven days significant changes were observed on the leaves of all treatment plants using checklist.

Morphological change
After 7 days of the exposure of plants by fungus (Aspergillus flavus) various morphological changes were observed. Dark brown blotches on the upper leaves surface turned black because of mold growth on them (Fig. 2) showed the prevalence of gray mold disease. After that, all leaves of plant wilted and died.Appearance of dark brown spots with slightly green edges which resulted in the dark brown dry foliage (Fig. 3) showed the presence of late blight disease. Greenishyellow spots which turned brown at the margins or tip of the leaves were observed which indicates the presence of leaf mold disease, a specific fungal disease (Fig. 4).

Endophytic effect on plant growth
Endophytes significantly reduced the growth of plants. It did not cause direct mortality of plants rather than prevalence of different diseases in plants were observed which resulted in causing mild to severe infections in plants demonstrating the pathogenicity of Aspergillus flavus(Table1). Differences in endophytic fungal effect in different treatment group tomato plants were observed (weakened or damaged leaves were affected at first), compared with the plants of control group which has shown no endophytic effects (Fig. 5).  The growth rate of conidia on exposed leaves was different and it was observed further. After 10 days there was no substantial further growth of Aspergillus flavus colonies.

Morphological and microscopic examination of recovered Aspergillus flavus colonies
The slide containing the fungal conidia grown by taking exposed leaves on PDA plates was microscopically observed and round shaped conidia were seen. It clearly indicated the presence of Aspergillus flavus conidia on exposed plant leaves. The conidial heads observed under microscope typically radiate longitudinally, conidiospores are un-colored, coarsely roughened, and approximately less than 1mm long. The samples were further identified for macroscopic and microscopic morphological characteristics by the experts. The severity level varied among different treatment plants. Moreover, this disease was more prevalent than late blight in all treatment plants. Severity level of gray mold, late blight and leaf mold disease in 10 7 , 10 6 , and 10 5 treatment plants is graphically presented (Fig. 6, 7, 8).  In present study, brown wilted leaves after 5 to 7 days of fungal inoculation showed dark brown blotches which turned black over time. Damaged or old leaves were found to be more affected. The leaves died after 7 days. These observed symptoms were similar to the symptoms of gray mold disease. Identification of the factors behind this effect was not clear. It has been reported that gray mold causes brown, water soaked spots or decay on leaves or petals. Tiny, almost translucent spots appear as the first symptoms. Infected terminals and stems may girdle and rot. Wilting of diseased tissues and death of infected seedlings occurs. Active, healthy tissues except petals are seldom infected directly. Most crops are attacked on the weakened (such as cutting stubs and tissue damaged by other pathogens), or injured, old, or dead parts. Primary limiting factor of this disease is moisture. Only after about 6 or more consecutive hours in contact with water, including free moisture from splashing, condensation or where relative humidity is higher than about 90%, Gray mold spores germinate and produce new infections. The optimum temperature for gray mold development is 16⁰C to 25⁰C [21]. It has also been reported in another study that characteristics circular or oval spots from yellowish to reddish brown in color is caused by infection of leaves. The spots dry out in dry weather and fungus stops spreading. When the weather is moist, the spots run together so that the whole leaf may be blighted. Stems also are attacked and when disease has progressed sufficiently the stem may break over at the point of infection [22]. After 7 to 10 days of leaf spraying some changes were observed in physical health of plant. Brown spots on leaves with slightly greenish boundary were present which was similar to the symptoms of late blight disease. The effect increased in high humidity. As it has been reported in recent study that late blight is serious fungal disease. The disease is detected by the first shown symptoms that are often present on lower leaves. . The root effect may be due to aflatoxin toxicity as it has been shown to inhibit root hair development in tobacco. Aspergillus flavus may also cause a mature peanuts rot in the soil. In cotton, cotton quality is affected by Aspergillus flavus by causing boll rot [28].Species of Aspergillus in the Aspergillus flavus group frequently causes mold of peanuts, Arachishypogaea L., and contamination with aflatoxins. When peanuts are stored under high humidity then it favors the growth of the fungus and this increases the potential for aflatoxins production [20]. Inoculation of exposed silks of two field-grown commercial dent cultivators 0,1,2, and 4 week after silk emergence resulted in infection and aflatoxins production [18].
In present study, Aspergillus flavus was found to colonize the tomato plant.
Although colonization was greatest in 10 7 treatment plants. The low rate of colonization was seen in 10 5 treatment plants. A significant change was observed in plant physical health when compared to control plant although no colonization was observed in control plants. As it has been reported that three entomopathogens Beauveria bassiana, Lecanicillium lecanii, and Aspergillus parasiticus were found to endophytically colonize a range of crop plants and could be isolated from inoculated leaves of cotton, bean, corn, tomato, pumpkin, and wheat. Although colonization of leaves was generally greatest in the most recently inoculated leaves, it consistently declined thereafter [29].

Conclusion
In agro-ecosystem Entomopathogens are one of the natural enemies of insect pests. Aspergillus spp. causes disease in a wide range of organisms, in plants, insects, and other animals. Our results show that Aspergillus flavus has parasitic attributes that is not specialized to a particular host. This study revealed significant data of the interaction between Aspergillus flavus and plants through which Aspergillus flavushinders plant growth and they cause impact on plant physiology. The symptoms related to late blight, leaf mold and gray mold disease were observed. This present work showed that Aspergillus flavus could infect tomato plant by contacting directly with conidia. A higher mortality rate was shown because of direct contact by leaf spraying. As it has been reported entomopathogenic; but the endophytic colonization of tomato plant leaves and tissues by Aspergillus flavus and its capacity to infect provides the basis for further investigation, on the virulence of the endophytic Aspergillus flavus.