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Section 1. Biology
Abdullaev Ikram, Doschanova Manzura, Rakhimbaeva Feruza, Matyaqubov Zafar, The Urgench State University, Urgench, Uzbekistan
Raina Ashok,
USDA-ARS-SRRC, New Orleans, Louisiana, United States
E-mail: [email protected]
Use of Beauveria tenella (Delacr.) Siem. as a microbial control agent against termites in Uzbekistan
Abstract: The fungus Beauveria tenella (Delacroix) Siemaschko was investigated as a microbial control agent against an insect pest species, the Turkestan termite, Anacanthotermes turkestanicus (Isoptera: Hodotermitidae). B. tenella was found to cause 100 % mortality of A. turkestanicus in 7 days as a wet preparation and 8 days as a dry preparation.
The tests showed that the biological effectiveness of propagules of B. tenella in experimental nests was 79.2, 78.5 and 96.5 % at concentration of 0.3, 0.5 and 1.0 g. per nest respectively.
Keywords: fungal pathogen, insect pest, microbial control.
Introduction
Termites are one of the most ancient groups of insects. They actively participate in the recycling of cellulosic material and in soil formation. There are approximately 2900 termite species nearly 160 of which are known to destroy wooden structures [7, 238]. Two of these termite species A. turkestanicus and A. ahngerianus, belonging to the genus Anacanthotermes (Isoptera: Hodotermitidae) inhabit Uzbekistan. Wood structures such as buildings, monuments of architecture and culture as well as wood in hydroelectric dams are subject to the destructive activity of termites [4, 60-64]. This problem is particularly serius in the southern Aral region of Uzbekistan.
Available methods of termite control, such as liquid termiti-cides and impregnation of wood with chemicals have not provided reliable long-term protection from termites in areas where Anacanthotermes species are found [8, 57-61; 11, 28-30]. A promising, but little studied, approach is the use of entomopathogenic microorganisms for the control of termite populations. This necessitates investigation of termites in this geographic region and development of control measures incorporating microbiological agents. Microorganisms and products of their metabolism are used against other insect pests; however, their use as control agents against termites is poorly developed [10, 419-428]. The interrelations between termites and microscopic fungi are multilateral, from symbiotic to antagonistic [12, 443-448].
The goal of this study was to evaluate selected potential microbial control agents for effectiveness against A. turkestanicus. The objectives included a determination of mortality of termites by individual fungal species, comparison of materials on which the candidate fungus can be efficiently harvested, and determining whether incubation time affects the potential of the fungus to kill termites.
Material and methods
Approximately 2000 termites were collected from Khoresm, Bukhara and Samarkand region of Uzbekistan and analyzed to
determine the mycobiota harbored by the termites. Whereas majority of the termites examined were workers. Other castes (nymphs, soldiers and alates) were also analyzed. The mycobiota of termites was obtained by placing dead termites into humidified chambers on agar plates. The termites selected for the analysis were washed with alcohol for 1 min and rinsed in sterile water prior to incubation. Following incubation at 24 °C for 10 days, the bacterial and fungal colonies that grew from the termites were replated onto Czapek's medium for further identification [5, 5-9; 14, 187].
The cultivation of B. tenella was conducted as follows: cellophane disks 12-15 cm. in diameter were boiled in distilled water for 15 min. and placed onto the surface of PDA. Inoculation was conducted by spraying 0.2 ml. of B. tenella water suspension onto the surface of the medium in one Petri dish. The fungus was incubated at 24 °C for 15-20 days. The fungus-covered disks were removed and air-dried. After a careful grinding of the fungus mass a powder-like fungal preparation was obtained, which was packed into 1 g capsules and sealed using a vacuum collector for storage of the fungus and application of the fungus as a dry preparation.
The concentration of water suspensions of fungal spores was calculated using a counting chamber and Goryaev's method [2, 293]. Calculations were made according to the formula: Ts = A x 250 x 10 3, where T is the titer of the suspension, and A is the average value of the number of spores in one large square. The inoculation of termites as a wet preparation was carried out by placing 10-20 individuals into a Petri dish which contained a disk of filter paper soaked with a 1 ml. suspension of 5 x 10 8 spores/ml in water. Filter paper disks soaked in water alone were used as the control. The experiment was replicated three times. The percent mortality of termites was calculated by Abbot's formula: X = (Mo - Mk)/(10 - Mk) x 100, where Mo is the number of dead termites in the experiment, and Mk is the number of dead termites in the control.
A strain of B. tenella, BD 85, had been isolated from the Moroccan locust and evaluated for virulence against termites in 1987. In preliminary tests this strain appeared to be more effective against the Turkestan termite, consequently we used this strain for all the bioassays.
The effect of the fungus on termites in the field was determined in four termite nests sufficiently apart. One of these nests was used as control. Six containers containing the dry fungal mass mixed with chopped sunflower stems were placed into the soil around each ofthe other three nests. Each of these nests received the fungus at one ofthe following concentrations; 0.3, 0.5 and 1.0 g. per nest. Six months after the treatment all the four nest were opened and the termites counted.
Data were analyzed using analysis of variance (ANOVA) and least significant difference (LSD) at P < 0.05 [3, 113]. All analyses were run using the SAS System Software.
Results
Termites of all castes, except alates, were found to harbor fungal pathogens. The most frequent cause of mycosis in the termites was the entomopathogen B. tenella. The occurrence of this entomopathogen in various lots of insects selected for the analysis ranged from 0.5 to 24%. Among other entomopathogens, we noted Aspergillus (A. flavus, A. oryzae and A. niger), the occurrence ofwhich in different lots of insects reached 11.5%. Species of the genus Penicillium were recorded on termites less frequently (0.9-5.9%). Single cases of isolation of the genera Fusarium, Alternaría and Cladosporium were also recorded. On a number of individuals we recorded a mixed infection, as we isolated B. tenella with Mucor sp.; B. tenella with Penicillium sp.; and, B. tenella with Alternaria sp. The mixed infection of termites with B. tenella and A. flavus was most frequent. Only sole representatives of the genus Penicillium were isolated from the ho-mogenate of termites, which suggests a poor mycobiota of the gut of termites and susceptibility of the fungal species to the food enzymes of termites [13, 13-20]. Thus, the pathogenic mycobiota of these termites was represented by a relatively small number of species.
For the selection of the optimal nutrient medium for the growth of B. tenella we tested the medium from a natural substrate - potato agar (PDA) and the medium of a certain mineral composition -Czapek's agar (Table 1).
Table 1. - The growth of biomass of fungus B. tenella in different nutrient media
Duration of cultivation (days) Biomass (mg.) of B. tenella from three Petri dishes
On PDA Czapek's medium
5 0.3 0.3
15 0.7 0.4
20 0.8 0.4
25 0.8 0.5
30 0.8 0.5
It was determined that PDA was preferable for obtaining a high fungal mass, the yield ofwhich was 1.5-2.0 times as high as on other agarized media (Czapek's medium) therefore, PDA was used in fUrther experiments both for the cultivation of the entomopathogen and for long-term storage of fungal cultures.
Growth of B.tenella on the food film resulted in a biomaterial yield of 0 %. The smallest amount of the fungal biomass was obtained at the use of the food filter. When we used different types of the filter paper, the obtained biomass was much lower than at the use of cellophane: 1.55 g., 1.70 g., 2.00 g. and 2.05 g., for dark-blue, white, blue filter paper and tracing paper, respectively, against 7.35 g. for the cellophane. The yield of the dry fungal mass (1.25 g.) during the growth on cellophane also exceeded other variants (Table 2).
Table 2. - The fungal weight of B. tenella and percent fungus yielded from separating membranes
Membrane Crude fungal weight (g.) Dry fungal weight (g.) Yield of biomaterial ( %)**
Food film, non-porous 0.00 0.00 00.0 C
Food filter 0.75 0.14 20.7 A
Filter paper (dark blue) 1.55 0.45 33.5 A
Filter paper (white) 1.70 0.45 28.3 A
Filter paper (blue)* 2.00 0.32 15.5 A
Tracing paper 2.05 0.32 15.0 A
Cellophane 7.35 1.25 17.3 A
Note:* — The blue, white and dark blue filter paper differed in pore size (n = 5); ** — Values with the same letter are not significantly different (P < 0.05).
It was also important to determine if the time of incubation affected the yield ofbiomaterial. When grown for 10 days the yield of dry fungal weight was 1.25 g., while at 15 days the yield was 1.30 g. (Table 3). The values were not significantly different, so these times of incubation did not affect the yield of biomaterial.
The amount offungal spores from 1 sq. cm. of culture constitutes, on average, 5.0 x 10 6; from the surface of one Petri dish it is possible to obtain 0.3 g. of preparation, which contains 7.5 x 10 8 spores.
Table 3. - Yield of B. tenella as a function of the time of incubation
Period of incubation (d.) Crude fungal weight (g.) Dry fungal weight (g.) Yield of biomaterial (%)
10 7.35 1.25 17.4 A*
15 8.35 1.30 15.7 A
Note:* — Values with the same letter are not significantly different (P< 0.05) (n = 10).
The development of mycoses in insects depends on the susceptibility of insects and the virulent traits of a fungus. It was experimentally established that the death of all experimental insects occurred within seven days after infection by the fungus B. tenella as a wet preparation; the highest number of dead termites was noted on days 6 and 7. In comparison, inoculation with A. flavus caused death in 76.6 % of termites in 10 days. Penicillium sp. caused 13.0 %; and Fusarium sp. caused 11.7 % mortality. Species of the genera Cladosporium and Alternaria caused death in only 7 and 4 % of termites, respectively. In the control group, 0-2 termites died each day.
The first signs of mycosis and death of termites in all variants were recorded on the third day of inoculation. With Mucor sp., the death of termites in experimental variants did not exceed the values of the control. This fungal species does not appear to have a pathogenic effect on the termites.
B. tenella was selected for further investigation based on the high rate of mortality it caused among termites. To obtain a dry fungal preparation we conducted a number of experiments on the identification of the composition of a nutrient medium, the type of the separating membrane, and the optimal incubation time for the entomopathogen. The entomopathogen B. tenella, like many other fungal species, grew well on various artificial nutrient media [9, 536-538].
The mortality of termites when exposed to the dry powder in comparison with water suspension of B. tenella spores was determined. The experimental data were recorded when death began on day 3. Both the dry preparation and the spores in water caused 100 % mortality of termites, but the dry fungal preparation took 24 hours longer, 8 days vs. 7 days (Table 4). The pathogen B. tenella was reiso-lated during the mycological analysis of the dead termites.
Table 4. - Effect of the dry and wet preparations of B. tenella on termite mortality
Days
3 4 5 6 7 8
Dry 10.0 A* 10.0 A 23.3 A 63.3 B 76.7 B 100.0 B
Wet 3.3 A 16.7 A 50.0 A 80.0 B 100.0 C 100.0 B
Control 3.3 A 3.3 A 3.3 A 6.7 A 6.7 A 10.0 A
Note: *— Mortality is expressed as mean percent. Values with the same letter are not significantly different (P < 0.05). Preparations were applied at a concentration of 5 x 106 spores/ml. (n = 3).
A test of baits containing B. tenella, which was placed into the soil around the termite nest, showed a sharp decline of termite numbers in the experimental nests six months later. In the control nest, the average number of termites was 17,200. In the treated nests the corresponding numbers were 5000, 3700 and 200 for 0.3, 0.5 and 1.0 g. B. tenella doses respectively. These results indicate a mortality of79.2, 78.5 and 96.5 %, respectively for the three treatment doses.
The mycological analysis of baits, which were subsequently removed from the soil, showed viable propagules of B. tenella, as the pieces of baits were overgrown by the test fungus. The propagules of B. tenella were recorded in the termite nests, and 2 % of the soil samples analyzed were infested with this fungus. A decline in the number of termites from neighboring nests where we did not place the fungus was also observed. This fact indicates that the fungus could have been distributed between colonies by termites.
Discussion
Microorganisms have been effectively applied for the microbial control of insect pests. However, the application of microorganisms for the specific control oftermites has been poorly utilized [17, 1-13; 10, 419-428]. Successful microbial control agents must compete with resident microbes and infection may result from stimulation of endemic pathogens or opportunists [12, 443-448]. Virulent isolates can be derived from a target insect infected in the field [6, 547-612]. Termites live in association with pathogenic microbes [12, 443-448]. Chemical termiticides may allow synergistic effects [15, 495-524].
The study of the mycobiota of termites inhabiting Khoresm and Samarkand provinces of Uzbekistan and the development of a method of reducing termite numbers with microbes was enabled by the determination of several factors. The mycological analysis of approximately 2000 individual termites from various castes showed that the most commonly observed fungal species were of the genera Beauveria and Aspergillus. In addition, species of the genera Penicillium, Fusarium, Alternaria and Cladosporium were recorded. Beauveria isolates have been previously shown to cause mortality to other species of termites [1, 538-543; 16; 18, 208-215].
Once a potential entomopathogen is isolated it is important to have an efficient method to propagate the microbe. An evaluation of materials on which to obtain fungal preparations without media contamination revealed that the optimal nutrient medium for the growth and storage of the fungal culture was PDA. Cellophane yielded the greatest dry fungal weight when used as a separating membrane. Both were porous materials that allowed the nutrients from the solid media surface to reach the fungal mass. The non-porous food film completely blocked the supply of nutrients which resulted in the inhibition of fungal growth, and proved unsuitable for this purpose. The cultivation of a fungus in controlled conditions on an appropriate separating membrane can allow harvesting of a media-free biomass of the pathogen. The material used as a separating membrane between the culture and the nutrient medium ideally provides a supply of nutrients from the substrate to the culture, and also prevents the medium from combining with the biopreparation when harvesting the fungal mass. Cellophane proved to have the most suitable qualities of the materials testes. The yield of fungal biomaterial did not vary significantly from 10 to 15 days incubation. Thus, it was preferable to cultivate B. tenella on PDA, cellophane can be used as a separating membrane between the fungal mycelium and the agar, and times of incubation of 10 and 15 days were equally effective. This method of cultivation of B. tenella provides approximately 18.1 % of the dry powder. In various lots this value ranged from 15.3 to 19.8 %. B. tenella caused 100 % mortality of termites in this study. Both dry and wet preparations were effective. The level of mortality caused by other fungal species, all isolated from termites in the course of this study, was significantly lower and did not exceed 40 %.
The use of baits containing 1 g. of B. tenella propagules under field conditions showed their high effectiveness in termite mortality six months after treatment. The ability of B. tenella to cause mortality of termites in both wet and dry compositions and to be harvested on membranes free of the growth medium makes it a prospective microbial control agent for A. turkestanicus.
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Berdiev Tolib Tursunniyazovich, Research Institute for Soil Science and Agro chemistry, researcher
E-mail: [email protected]
Agro technologies increasing the productivity of irrigated soils in the desert zone of Uzbekistan
Abstract: The results of research aimed to improving soil fertility and crop yields in the system "cotton-winter wheat" in accordance with the agricultural technologies developed by our institute, related to soils of the desert zone are given. The ways of increasing the amount of humus, nutrients in the soil and productivity of cotton and winter wheat are demonstrated.
Keywords: soil fertility; fertilizer; enrichment of the soil; organic substances; crop yields; the carbon content of humus.
In recent years in the country's major irrigated areas there is a tendency of reducing the content of humus, the nutrients available to plants, occurrence of negative phenomena as degradation, dehumidification, erosion, salinization, compaction, etc., which ultimately leads to poor land quality and reduction of soil fertility. A prevention of these negative phenomena is possible by land reclamation and the introduction of agricultural technologies to improve all the basic properties of the soil, enriching it with organic matter and the elements of plant nutrition.
On the basis of studies at period of 2003-2010, we developed agricultural technologies applicable to soils of serozem zone, aimed to enriching the soil with organic matter, increasing soil fertility and yield of crops.
This agrotechnology includes a cultivation of major crops in system "cotton-winter wheat" and their consequent alternation with repeated crops — corn combined with bean (mung bean, soybean) and intermediate crops (oats, canola) with obligatory introduction of high amounts oforganic fertilizers-manure from different origin, organo-mineral fertilizers, composts from local raw materials on the basis of manure. This reduces the rate of applied mineral fertilizers 1.5-2 times.
The same agrotechnology, with some modifications, developed in relation to takyr-meadow and serozem-meadow soils of the desert zone in several farms of Surkhandarya and Kashkadarya regions of the republic in 2008-2011.
The paper presents the results of research on the effect of applied agricultural technology on selected fertility indicators of irrigated takyr-meadow soils and productivity of crops (cotton, winter wheat), conducted in 2009-2011 on the territory of the farm of Angor district of Surkhandarya region.
Research methodology
Field experiments with the cultivation of major, repetitive and intermediate crops conducted by Soyuz NIHI (All-union research institute of cotton science) methodology. Soil analyzes were performed according to standard methods described in the manuals of Soyuz NIHI (1977) and E. V. Arinushkina (1970).
Experiments with cotton, winter wheat and other crops were placed in 5 cases, repeated 3 times. The size of the plot — 72 m2, the total area — 1080 m2.
Results
The results of the soil analysis on change the content of total humus in three years of experiments in cotton cultivation (SpringFall 2009), winter wheat (fall 2009 -summer 2010), sowing repeated crops (Summer-Fall 2010), intermediate (Fall 2010-Spring 2011) and re-cotton (spring-fall 2011) showed a significant increase of its content in the variants, which used the proposed agro technology with the introduction of various norms of manure with reduced 1.5-2 times the norm of mineral fertilizers, as well as microbiological preparation (MERS).
Thus, in the control variant without fertilizers from spring 2009 to the autumn 2011 there is decrease in the carbon content of humus in the upper arable (0-30 cm.) and subsurface (30-50 cm.) soil horizons at 0.095 %, which is equal to 7.64 t/ha. In variant with introduction only complete the norms of mineral fertilizers during this period was an increase in these layers of soil carbon content of organic substances in the 0.097 % and 0.050 %, respectively. This is for the 0-50 cm. soil layer is 5.88 t/ha. Such an increase in the carbon content oforganic matter was due to the better development of the plants due mineral fertilizers and more decayed root and crop debris.
The variants of the experiment, which used the proposed agricultural technology with application of organic fertilizer (manure) at the rate of 20 and 40 t/ha annually for three years and at a low of 1.5 and 2 times the rate of mineral fertilizers, in the 0-50 cm. soil layer was an increase amounts of organic substance on the 0.177 % and 0.239 %, which is equal to 7.08 and 9.56 t/ha, respectively (Fig. 1). These values are higher than the control group with mineral fertilizers by 1.2 and 3.68 t/ha. In variant of the experiment which used a microbiological preparation at rate 100 ml/ha with 1.5 times the low rate of fertilizers, increasing the amount of organic matter in the 0-50 cm. layer was 0.132 % or 5.28 t/ha, which is close to control variant which used the full rate of fertilizer.
