The parasitic and lethal effects of Trichoderma longibrachiatum against Heterodera avenae

Biological Control 72 (2014) 1–8

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The parasitic and lethal effects of Trichoderma longibrachiatum against Heterodera avenae Shuwu Zhang a,c,d,e, Yantai Gan b,f, Bingliang Xu a,c,d,e,⇑, Yingyu Xue a,c,d,e a

College of Grassland Science, Gansu Agricultural University, Lanzhou 730070, China Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2, Canada c Key Laboratory of Grassland Ecosystems, the Ministry of Education of China, Lanzhou 730070, China d Pratacultural Engineering Laboratory of Gansu Province, Lanzhou 730070, China e Sino-U.S. Centers for Grazingland Ecosystems Sustainability, Lanzhou 730070, China f Gansu Provincial Key Lab of Arid Land Crop Science, Lanzhou 730070, China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 T. longibrachiatum was found for the

first time to be an agent against H. avenae.  The best T. longibrachiatum concentration against H. avenae was at 1.5  108 spores/ml.  The parasitic and lethal effects of T. longibrachiatum were >91% after 18 days.  Chitinase activity was maximized 4 days after inoculation of H. avenae cysts.  Main mechanism of T. longibrachiatum against H. avenae was via increased chitinase.

a r t i c l e

i n f o

Article history: Received 28 April 2013 Accepted 26 January 2014 Available online 2 February 2014 Keywords: Trichoderma longibrachiatum Heterodera avenae Parasitic and lethal effects Biological control

a b s t r a c t Heterodera avenae is a devastating plant pathogen that causes significant yield losses in many crops, but there is a lack of scientific information whether this pathogen can be controlled effectively using biocontrol agents. Here we determined the parasitic and lethal effects of Trichoderma longibrachiatum against H. avenae and the possible mechanism involved in this action. Both in vitro and greenhouse experiments were conducted. In vitro, T. longibrachiatum at the concentrations of 1.5  104 to 1.5  108 spores per ml had a strong parasitic and lethal effect on the cysts of H. avenae, with the concentration of 1.5  108 spores per ml having >90% parasitism 18 days after treatments. In greenhouse, T. longibrachiatum inoculation decreased H. avenae infection in wheat (Triticum aestivum) significantly. Observations with microscopes revealed that after mutual recognition with cysts, the spore of T. longibrachiatum germinated with a large number of hyphae, and reproduced rapidly on the surface of cysts. Meanwhile, the cysts surface became uneven, with some cysts producing vacuoles, and the others splitting. Finally the cysts were dissolved by the metabolite of T. longibrachiatum. Chitinase activity increased in the culture filtrates of T. longibrachiatum and reached the maximum 4 days after inoculation in the medium supplemented with colloidal chitin (1.02 U/min per ml) and nematode cysts (0.78 U/min per ml). The parasitism and inhibition of cysts through the increased extracellular chitinase activity serves as the main mechanism with

⇑ Corresponding author at: College of Grassland Science, Gansu Agricultural University, Lanzhou 730070, China. E-mail address: [email protected] (B. Xu). http://dx.doi.org/10.1016/j.biocontrol.2014.01.009 1049-9644/Ó 2014 Elsevier Inc. All rights reserved.

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which T. longibrachiatum against H. avenae. In conclusion, T. longibrachiatum has a great potential to be used as a biocontrol agent against H. avenae. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Soil-borne and plant parasitic nematodes are the most important biotic constraint limiting the production of wheat (Triticum aestivum L.) crops worldwide. Since the cereal cyst nematode (Heterodera avenae) was first found in northern New South Wales, Australia in the early 1980’s (Southwell and McLeod, 1981), several other nematodes such as H. avenae, Heterodera filipjevi and Heterodera latipons have been found in other regions of the world. In some countries/regions, nematodes cause significant economic losses in cereal crops, especially when crops are grown under low rainfall and poor soil nutrition conditions (Barker and Noe, 1987; Nicol and Rivoal, 2008). In India and Rajasthan, H. avenae causes ‘‘Molya’’ disease on wheat and barley (Hordeum vulgare L.) and yield losses up to 47% in wheat and 87% in barley (Evans et al., 1993). In recent years, H. avenae has become a serious problem in China (Nicol et al., 2007; Li et al., 2010). Since it was first reported in Hubei Province in 1989, the nematode has been consecutively found in Hebei, Henan, Beijing, Shanxi, Inner Mongolia, Qinghai, Hubei, and Anhui, where millions of hectares of wheat fields are infected annually, with yield losses up to 50% (Peng et al., 2009; Yuan et al., 2010; Riley et al., 2010), and in many cases it decreases grain quality (Riley et al., 2010). Furthermore, H. avenae can be harmful to soil Rhizoctonia solani, leading to crop seedling rot and a variety of other fungal diseases, such as root rot caused by Phytophthora spp. (Peng et al., 2009). A number of strategies have been developed to control nematodes in agriculture, including the use of chemical and biological nematicides (El-alfy and Schlenk, 2002). Some of the chemical nematicides, such as Carbofuran, Etho-prophos and Miral, are effective in controlling H. avenae, but those nematicides often have a high level of toxic residues in the soil, with a high possibility of killing beneficial microorganisms (Sergio, 2011). Moreover, the application of nematicides may bring toxicity to human, animal, and the environment. The residues are difficult to degrade in soils, and they may pollute groundwater and environments (Jatala, 1986). In view of the limitation of chemical control, there is a growing incentive to develop environment-friendly products. Thus, a great number of biological control microorganisms have been screened and researched, including fungi (Crump et al., 1983; Chen and Dickson, 1996), bacteria (Becker et al., 1988), ray fungi, and protozoon (Chen and Liu, 2005; Sun et al., 2006). Some biological control products like Paecilomyces lilacinus and Pochonia chlamydosporia have been commercially used in certain areas (Leij De et al., 1992). However, in the complex of the soil environment, there exists an inhibitory effect on the microbes, thus limiting the effectiveness of microbial agents. Trichoderma spp. is a class of soil-borne fungi, serving as an antagonist to some soil-borne pathogens, such as Rhizoctonia, Sclerotium, Fusarium, Pythium (Deng et al., 2007). Among the antagonisms, Trichoderma harzianum (T. hatzianum) and Trichoderma viride (T. viride) have been studied in details (Gary, 1996; Louzada, 2009). Some of those antagonisms have been used in controlling rice sheath blight with the commercialized Trichoderma formulation in some countries (Baker, 1991). The strain of T. harzianum T22 (Topshield) has been used in the United States, and the strain of T. harzianum T39 (Trichodex) has been used in Israel (Elad, 2000; Alessandro et al., 2012).

Trichoderma longibrachiatum is a well-known bio-control agent against several plant pathogens without environmentally hazardous effects, but little information is available regarding the parasitic and inhibitory effects on the cysts of H. avenae. The mechanisms of T. longibrachiatum against nematodes are essentially unknown. Therefore, the present study was to (i) evaluate the ability and effectiveness of T. longibrachiatum in the control of H. avenae, and (ii) determine the probable mechanism with which T. longibrachiatum against cereal cyst nematode H. avenae. 2. Materials and methods 2.1. Nematode inoculum preparation and plant material Soil samples were collected from a wheat field at each of the three sites – Dingxi, Wuwei and Yongdeng (Gansu Province, China). A single cyst of H. avenae was used to establish a population on wheat (cv., Yongliang 4, susceptible to H. avenae) for the experiment. Heterodera avenae cysts were obtained using ‘‘Flotation separation’’ method as described by Long et al. (2012). The isolated H. avenae cysts were immersed in 1% NaOCl for 1 min, and then were gently washed with tap water to remove NaOCl. Experiments were carried out in a greenhouse with constant temperature of 25 °C ± 0.5, supplemental day/night lighting of 16/8 h, and relative humidity of 65%. 2.2. Fungal inoculum preparation Trichoderma longibrachiatum, obtained from Gansu Agricultural University Plant Pathology Laboratory, was cultured on potato dextrose agar (PDA) in Petri dishes for 6 days at 25 °C. The conidia suspension of T. longibrachiatum was then prepared by flooding the dishes with 3 ml of sterilized distilled water, the agar surface was gently scraped with sterile glass rods, and the suspension collected in a sterile 250 ml beakers. The suspension was then adjusted to 50 ml, and mixed using a hand mixer to separate and disperse the conidia before assessing conidia density with a haemocytometer, and adjusted to 1.5  108 conidia per ml. Each erlenmeyer flask (150 ml) contains 60 ml of sterilized liquid culture medium of potato dextrose broth (PDB) and inoculated with 1 ml of the conidia suspension of T. longibrachiatum inoculum (1.5  108 spores/ml) under aseptic conditions, and shaken at 120 rpm and 30 °C for 9 days. Nine days after incubation, the purified fungi were used to produce spore suspension for inoculation. A drop of Tween-80 was added to PDB that had been inoculated with the conidia suspension of T. longibrachiatum, and the spores of T. longibrachiatum were shocked off in sterile water. The spore suspension of T. longibrachiatum was filtered through a Whattman Paper No. 3 filter and followed by filtration through 0.22 mm Millipore membranes. It was then adjusted to 1.5  108, 1.5  107, 1.5  106, 1.5  105 and 1.5  104 spores per ml and stored at 4 °C. 2.3. Effect of different concentrations of T. longibrachiatum on the cysts of H. avenae in vitro Ten surface sterilized cysts with a similar physical size were placed in each Petri dish (6 cm in diameter and sterilized), and

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then 5 ml concentrations (1.5  108 spores/ml) of T. longibrachiatum and sterile supernatant of PDB were added to each Petri dish, and then were incubated at 25 °C. This experiment was replicated three times. Sterile supernatant of PDB was used as control. The process of infection was observed at 2 days intervals. Ten surface sterilized cysts with a similar physical size were placed in each Petri dish (6 cm in diameter and sterilized), and then 5 ml of each of the five different concentrations (1.5  108, 1.5  107, 1.5  106, 1.5  105 and 1.5  104 spores/ml) of T. longibrachiatum, along with a control (T. longibrachiatum was replaced with sterile supernatant of PDB). These Petri dishes were incubated at 25 °C. This experiment had six treatments (including the control) with three replicates. Hatching and parasitism were observed 10 days after incubation. The percentages of inhibition and parasitism were calculated according to the method of Gao et al. (1998), as follows:

PPCð%Þ ¼ ðNCPET=TNTCÞ  100;

ð1Þ

where PPC represents the percentages of parasitism on cysts, NCPET the number of cysts parasitized in each treatment, and TNTC the total number of test cysts.

RPICHð%Þ ¼ ðNNHCG-NNHETÞ=NNHCG  100;

ð2Þ

where RPICH represents relative percentages of inhibition of cysts hatching, NNHCG the number of nematodes hatched in the control group, and NNHET the number of nematodes hatched in each treatment. 2.4. Biocontrol experiments Wheat seeds that were surface sterilized with 1% sodium hypochlorite for 5 min were sown in pots (10 cm diameter, 30 cm in height) containing sterile medium (a mixture of field soil and sand 1:1 v/v). Each pot had 10 seedlings and they were fertilized with liquid nutrient solution every two days after seedling emergence. Wheat seedlings at the two-leaf stage were inoculated with T. longibrachiatum and two days after were inoculated with 150 cysts in each pot. The experiment included six treatments, including five T. longibrachiatum concentrations at 1.5  108, 1.5  107, 1.5  106, 1.5  105 and 1.5  104 spores/ml, respectively, and a control (inoculated with cysts but not with T. longibrachiatum which was replaced with sterile supernatant of PDB). Holes with a pencil point size were made around the seedlings and 20 ml of the T. longibrachiatum concentration was applied per pot. The inoculated seedlings were grown at 25 °C, with supplemental day/night lighting of 16/8 h, and relative humidity of 65%. The pots were irrigated with sterile distilled water. Forty days after seedling inoculation, nematode damage (number of cysts, females and juveniles) were recorded and evaluated. 2.5. Evaluation of different concentrations of T. longibrachiatum for chitinase production Seven grams of dry wheat bran was placed in a 250 ml Erlenmeyer flask and was supplemented with 3 ml of salt solution containing 0.5% NH4NO3, 0.2% KH2PO4, 0.1% NaCl, and 0.1% MgSO4 + 7H2O (Sahebani and Hadavi, 2008). The initial moisture level in the substrate was adjusted by adding adequate amounts of sterile distilled water. The substrate was mixed thoroughly and autoclaved for 20 min at 121 °C and cooled to room temperature before inoculation. The sterilized solid substrate medium was inoculated with 1 ml of different concentrations of T. longibrachiatum spore inoculum (1.5  104–108 spores/ml) under aseptic conditions. The contents were mixed thoroughly and the flasks were placed in an incubator at 30 °C for 10 days. Flasks were removed

3

every 24 h and the enzyme extraction and chitinase assay were performed as described below. An adequate amount of distilled water containing 0.1% of Tween-80 was added to the substrate to get a total extraction volume of 100 ml. The contents were mixed thoroughly by keeping the flasks on a rotary shaker at 150 rpm for 30 min. The mixture was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected and used for chitinase assay. This experiment included six treatments (including one control) with six replicates. The activity of chitinase production was observed at 1 day intervals. The activity of chitinase was assayed by measuring the release of reducing saccharides from colloidal chitin as follows (Miller, 1959): a reaction mixture containing 1 ml of culture supernatant, 0.3 ml of 1 M sodium acetate buffer, pH 4.7, and 0.2 ml of colloidal chitin (sterilized water was used as control) were incubated at 50 °C for 1 h and then centrifuged at 10, 000 rpm for 5 min at 4 °C. After centrifugation, an aliquot of 0.75 ml of the supernatant, 0.25 ml of 1% solution of dinitrosalycilic acid in 0.7 M NaOH, and 0.1 ml of 10 M NaOH were mixed in 1.5 ml Eppendorf tubes and heated at 100 °C for 5 min. Absorbance of the reaction mixture at 530 nm was measured after cooling to room temperature using UV spectrophotometer. A calibration curve with N-acetyl-D-glucosamine (NAGA) as a standard was used to determine the reducing saccharides concentration. Under the assay conditions, a linear correlation between A530 and NAGA concentration was found in the interval of 40–800 mg/ml of NAGA. One unit (U) of the chitinase activity is defined as the amount of enzyme that is required to release 1 mmol of NAGA per minute from 0.5% of dry colloidal chitin solution under assay conditions. 2.6. Nematode cysts as a chitinase inducer Erlenmeyer flasks (500 ml) containing 150 ml of growth medium (0.03% K2HPO4, 0.03% MgSO4 + 7H2O, 0.03% NaCl, 0.02% yeast extract, and 1% (w/v) glass wool) were sterilized by autoclaving at 121 °C for 30 min. Sterile nematode cysts (10 cysts) as the main source of N and C and 1 ml of different concentrations of T. longibrachiatum spore inoculum (1.5  104 to 108 spores/ml) (the growth medium inoculated with T. longibrachiatum but not with cysts was used as control) was used as inoculums. These were incubated at 30 °C in dark for 10 days. Culture medium was filtered through a Whattman Paper No. 3 filter followed by filtration through 0.22 mm Millipore membranes. The filtrate obtained was analyzed for chitinase activity. This experiment had six treatments (including one control) and was replicated six times. The activity of chitinase production was observed at 1 day intervals. 2.7. Data analysis The data were subject to one-way analysis of variance (ANOVA) using SPSS package (SPSS V16.0, SPSS Ltd., Chicago, IL). Fisher’s least significant differences (LSD) were used to determine the significant differences between treatments at P 6 0.05. 3. Results 3.1. Microscopic observations of the process of H. avenae cysts infestation by T. longibrachiatum in vitro The spore of T. longibrachiatum had a strong inhibitory and parasitic effect on the cysts hatching, compared to the control. Microscopic examinations revealed that the spore of T. longibrachiatum adsorbed or parasitized on the cyst surface 10 days after treatments, and a large number of spores germinated and parasitized

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Fig. 1. The morphological characteristics of the cysts of H. avenae infected by the spore suspension of T. longibrachiatum in vitro, where (A) and (B) the cysts were absorbed and parasitized by the spore of T. longibrachiatum; (C) and (D) the cysts were parasitized by the mycelium of T. longibrachiatum; (E) the cyst with engendered vacuole and deformity; (F) and (G) the cyst was split and parasitized by the spore suspension of T. longibrachiatum; (H) the cyst was dissolved by the metabolites of T. longibrachiatum; and (I) the cyst in the control group.

on the surface of the cyst (Fig. 1A and B). Two days after treatments, the spores started reproduction on the surface of the cysts, and a large number of hyphae germinated and penetrated into the cyst shell, with the whole cyst surrounded by dense mycelium (Fig. 1C and D). At the same time, the cyst began to de-formatted; some cysts changed their color from brown to tan, and the cyst surface became uneven, and some cysts produced vacuoles and appeared malformed 14 days after treatments (Fig. 1E). Even some cysts were split up (Fig. 1F and G) 16 days after the treatments, and finally the cyst was dissolved by the metabolites of T. longibrachiatum (Fig. 1H) 18 days after the treatments. There was no parasitic mycelium in the control group (Fig. 1I). 3.2. Effects of different concentrations of T. longibrachiatum on the cysts of H. avenae in vitro Trichoderma longibrachiatum at the various concentrations all showed a significant inhibitory and parasitic effect on the cysts of H. avenae hatching, and the increased concentrations of T. longibrachiatum increased the inhibitory and parasitic effect. In contrast, the control (inoculated with cysts but not with T. longibrachiatum which was replaced with sterile supernatant of PDB) had no inhibitory and parasitic effect on the cysts. A significantly higher percentage of cysts was parasitized by T. longibrachiatum in the treatment groups with the different concentrations compared to the control (Table 1). The percentage of parasitism increased with the increased concentration from 1.5  104 (averaging 40%) to 1.5  108 spores/ml (averaging 78%). The length of the treatments also had a significant effect on the cysts with the parasitism of cysts increasing from an average of 43% at 10 days after incubation to 81% at 18 days after incubation. The percentage of parasitism reached 93% at day 18 after the treatments with the highest T. longibrachiatum concentration at 1.5  108 spores/ml. There was no fungus parasitized in the control group.

Table 1 Effects of different concentrations of T. longibrachiatum on parasitism of H. avenae cysts in vitro, measured at different days (d) after incubation. Concentration (spores/ml)

Days after incubation (d) 10

1.5  108 1.5  107 1.5  106 1.5  105 1.5  104 Control

12

14

Percentages of parasitism (%) 63.3 a 70.0 a 73.3 a 53.3 a 60.0 ab 66.7 ab 46.7 ab 53.3 b 60.0 b 30.0 bc 33.3 c 43.3 c 23.3 c 30.0 c 33.3 c 0.0 d 0.0 d 0.0 d

16 90.0 86.7 76.7 66.7 50.0 0.0

18 a ab c c c d

93.3 90.0 83.3 76.7 63.3 0.0

a ab bc cd d e

Means in a column followed by different letters are significantly different based on Fisher’s LSD test at P 6 0.05.

Compared to the control, the percent inhibition of cysts hatching increased with the increase of T. longibrachiatum concentrations (Table 2). The relative percentages of inhibition reached the highest 10 days after the treatments and again 18 days after treatments. These results indicate that T. longibrachiatum not only reduces cysts hatching but also causes cysts weakness and mortality. 3.3. Effects of T. longibrachiatum inoculation on H. avenae quantity in soil and on roots Under in vivo conditions, T. longibrachiatum had a significant effect on H. avenae cyst hatching, female and juvenile development (Table 3). Wheat seedlings inoculated with the different concentrations of T. longibrachiatum significantly decreased the number of H. avenae cysts and females, and suppressed juvenile development on both the soil and crop roots, when compared with the control. With the increase of T. longibrachiatum concentration from 1.5  104 to 1.5  108 spores/ml, the control efficacy (i.e., percent

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S. Zhang et al. / Biological Control 72 (2014) 1–8 Table 2 Effects of different concentrations of T. longibrachiatum on the cyst of H. avenae hatching in vitro, measured at different days (d) after incubation. Concentration (spores/ml)

Days after incubation (d) 10

1.5  108 1.5  107 1.5  106 1.5  105 1.5  104 Control

12

14

Percent inhibition (%) relative to the control 93.6 a 91.0 a 83.2 b 79.7 b 70.4 b 65.4 b 60.0 c 54.2 c 52.0 d 48.5 d

91.0 79.6 66.3 53.6 48.4

___

___

___

16 a b b c d

90.8 79.5 65.8 52.5 48.2

18 a b b c d

91.1 80.0 67.3 54.4 50.3

___

a b b c d

___

Means in a column followed by different letters are significantly different based on Fisher’s LSD test at P 6 0.05.

Table 3 Quantity of H. avenae in the soil and wheat roots measured 40 days after the inoculation of different concentrations of T. longibrachiatum in greenhouse experiments. Concentration (spores/ml)

8

1.5  10 1.5  107 1.5  106 1.5  105 1.5  104 Control

Number of H. avenae in soil and roots Cysts per 200 g of soil

Juveniles per 200 g of soil

Juveniles per 2 g of fresh root

Females per 2 g of fresh root

18.7 25.3 29.7 38.7 52.3 125.7

82.7 124.0 194.3 348.7 515.7 1149.3

25.3 44.3 58.7 68.0 77.0 130.0

5.0 11.0 17.0 23.3 29.0 49.7

e d d c b a

f e d c b a

e d d c b a

f e d c b a

Means in a column followed by different letters are significantly different based on Fisher’s LSD test at P 6 0.05. Control represents seedlings inoculated with cysts but not with T. longibrachiatum.

decreases of nematodes in the T. longibrachiatum treatments compared to the control) increased from 42 to 90% for females on the roots, from 41 to 81% for juveniles on the roots, from 58 to 85% for cysts in the soil, and from 45 to 93% for juveniles in the soil, as compared to the control. 3.4. Chitinase production and activity Both colloidal chitin (Fig. 2A) and nematode cysts (Fig. 2B) induced chitinase activity and the production. At a given day after treatments, the increased T. longibrachiatum concentrations increased the activity of chitinase significantly. In liquid minimal medium supplemented with colloidal chitin, the maximum chitinase production was 1.02 U/min per ml occurring at the 4th day after treatments, and thereafter the chitinase production declined slowly (Fig. 2A). In liquid minimal medium supplemented with nematode cysts, the maximum chitinase was 0.78 U/min per ml which occurred at the 4th day after treatments, and thereafter declined slowly (Fig. 2B). At any a day after treatments, the control had the lowest chitinase activity and the production regardless of the medium used. 4. Discussion Plant parasitic nematodes are one of the most important pathogens in crops. With the global warming and the continuous increase of intensive cropping, nematodes cause significant damages to many crop species (Gao et al., 1998). It is imperative to develop efficient and environment-friendly bio-control agents which can be used to prevent or minimize plant nematode diseases. Our results indicate that T. longibrachiatum has a great potential to become a bio-control agent against the cysts of H. avenae. In vitro, the different concentrations of T. longibrachiatum not only had a strong inhibitory and parasitic effect on the cysts, but also caused cysts weakness and mortality. Other researchers have attempted to use Trichoderma as a bio-control agent against plant-parasitic nematodes (Reddy et al., 1996; Rao et al., 1998). Direct interactions

between T. harzianum and the potato cyst nematode Globodera rostochiensis have been demonstrated in vitro (Saifullah and Thomas, 1996). In recent years, P. lilacinus and P. chlamydosporia have been used as nematode bio-control agents (Hahn, 1990), but many clinical trials indicate that P. lilacinus can infect human or animal skins, tissues and organs (Rockhill and Klein, 1980; Orth et al., 1996). Usually, P. lilacinus only parasitizes on nematode eggs and the infection rates are related to the length of time when it contacts with eggs (Bonants et al., 1995; Oclarit and Cumagun, 2009). Pochonia chlamydosporia is a common parasite of cystnematode females and eggs (Kerry and Crump, 1977; Vinduska, 1982), but the ability to colonize nematodes varies greatly (Bourne et al., 1996). Our experiments showed that the effect of T. longibrachiatum on the cysts was highly effective. Compared to the control, the different concentrations of T. longibrachiatum had higher percentages of parasitism and inhibition on cysts hatching. Other researchers have reported the effects of T. viride at the different concentrations on the hatching of egg masses and individual eggs and juveniles of Meloidogyne halpa (Liu et al., 2007). Also, nematophagous fungi have been reported to have the ability to infect or trap nematodes (Huang et al., 2004). The egg-parasitic fungi Paecilomyces spp. (Khan et al., 2004) and Pochonia spp. (Tikhonov et al., 2002) also infect nematode eggs and repress the hatching of juveniles, thereby reducing nematode populations. However, there has been little research reported on the cysts of H. avenae. Our greenhouse experiments showed that T. longibrachiatum significantly inhibited cysts and females of H. avenae, and suppressed the development of juveniles. In studies with other fungi, Khan et al. (2004) found that T. asperellum T-16 suppressed second stage juvenile of root-knot nematode densities in crop roots with a control efficacy up to 80%, and that T. brevicompactum T-3 suppressed egg production by as much as 86% compared to the control. Those authors also showed that the number of H. avenae cysts was reduced by 60% in barley crop when a combination of P. lilacinus and Monacrosporium lysipagum was applied. For the first time, we assessed the potential of T. longibrachiatum in parasitizing the cysts of H. avenae and evaluated the

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Fig. 2. The activity of chitinase in the culture filtrates with different concentrations of T. longibrachiatum (along with the control) growing in liquid media induced by (A) Colloidal chitin and (B) Nematode cysts. Small bars represent the standard errors of the means.

possible mechanism responsible for the control efficiency. This was done through the observation of the process of infection under microscopes and the evaluation of T. longibrachiatum for chitinase production in vitro. Microscope observations revealed that parasitism and some enzymes involved in the process were probably the important mode of action and one of the initial steps of this process against the cysts of H. avenae hatching in vitro. After the mutual recognition with the cysts, the spore of T. longibrachiatum germinated a large number of hyphae, and reproduced on the surface of cysts. Furthermore, we observed that the cyst surface became uneven, and some cysts produced vacuoles, even some cysts were split and finally dissolved by the metabolites of T. longibrachiatum. It is possible that T. longibrachiatum produces a kind of enzyme (such as chitinases) that causes the physiological disorder of cysts (Khambay et al., 2000; Sharon et al., 2007; Gortari and Hours, 2008; Pau et al., 2012). Also, it is possible that dissolved cysts bodies severely affect their physical vitality. Various mechanisms have been suggested for the bio-control activity of Trichoderma against phytopathogenic fungi, including competition, antibiosis, parasitism, enzymatic hydrolysis and systemic induced resistance (Chet et al., 1997; Harman et al., 2004). Sharon et al. (2001) showed direct parasitism of T. harzianum (T-203) on M. javanica in vitro, where extracellular protease enzyme was secreted by the fungus, but the percentage of direct parasitism was low. Larito et al. (1993) found that T. harzianum took place in the soil, out of plant roots or in the cortex tissue but they had no direct relationship

with nematode in the host tissue. Our results suggest that T. longibrachiatum causes direct and indirect effects on nematode cysts, and it can effectively reduce cysts hatching and activity. In our study, the evaluation of chitinase production under the different concentrations of T. longibrachiatum suggests that parasitism and inhibition of cysts hatching is most likely through the increase of chitinase in the control of H. avenae. Meanwhile, the cysts can significantly induce chitinase and increase its amount with the increased concentrations of T. longibrachiatum. It is well known that chitinases are required for hyphal growth, and this kind of inducible enzyme can catalyze chitin, an important component in fungal cell wall, nematode and insect eggshell and insect cuticle (Takaya and Yamazaki, 1998). Our results also showed that the proportion of infected nematode cysts increased concurrently with the enhancement of chitinase activity. Both proteolytic and chitinolytic enzymes may be required to disrupt the eggshell (Tikhonov et al., 2002; Khan et al., 2004), but chitinolytic capacity is the most important activity on the eggshells (Morton et al., 2004). Trichoderma longibrachiatum like other nematophagous fungi must be able to produce extracellular chitinase and protease enzymes. It is also possible that other lytic enzymes are involved in cysts penetration. Our study clearly demonstrates that T. longibrachiatum is effective in the prevention and control of H. avenae in vitro and vivo. However, the study on T. longibrachiatum is still in its infancy, and there are many issues that need to be addressed in the future

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studies, such as the effect of bio-control agents on nematodes eggs, second stage juveniles of H. avenae, and possible interactions with other extracellular enzymes. Also, there is a need to determine the safety of this bio-control agent to human, animals and plants, and the persistence of control efficacy under different environmental conditions, as well as the target nematode species. 5. Conclusion The results of our study suggest that T. longibrachiatum can be used as a bio-control agent for the management of H. avenae on selected crop species and it can be highly effective in some particular circumstances. Direct parasitism of cysts through the increases of extracellular chitinase activity is the main inhibition mechanism responsible for T. longibrachiatum against the cysts of H. avenae hatching. More research is suggested to determine the safety to human, animals, and plants, and the use efficacy in controlling target nematode species. Acknowledgments This work was supported by Plant Protection Department of Gansu Agricultural University; Key Laboratory of Grassland Ecosystems, the Ministry of Education of China; Sino-U.S. Centers for Grazingland Ecosystems Sustainability; Gansu Hall of Province Farming Herd Biology Technology and Project of Education Department of Gansu Province; Grassland Ecological System of Ministry of Education Ministry Key Laboratory Project (CY-GG-2006-013); Gansu Hall of Province Farming Herd Biology Technology (GNSW-2009-04) and Project of Education Department of Gansu Province (042-03). References Alessandro, V., Gabriella, C., Ivana, C., Dalia, A., Giancarlo, P., 2012. Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa. Biocontrol 57, 687–696. Barker, K.R., Noe, J.P., 1987. Establishing and using threshold population levels. In: Vistas on Nematology. Society of Nematologists, Maryland, pp. 75–81. Baker, R., 1991. Diversity in biological control. Crop Prot. 10, 85–94. Becker, J.O., Zavaleta, M.E., Colbert, S.F., Schroth, M.N., Weinhold, A.R., Hancock, J.G., Vangundy, S.D., 1988. Effects of rhizobacteria on root knot nematodes and gall formation. Phytopathology 78, 1466–1469. Bonants, P.J.M., Fitters, P.F.L., Thijs, H., Den Belder, E., Waalwijk, C., Henfling, J.W.D.M., 1995. A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs. Microbiology 141, 775–784. Bourne, J.M., Kerry, B.R., Leij De, F.A.A.M., 1996. The importance of the host plant on the interaction of root knot nematodes and the nematophous fungus, Pochonia chlamydosporia goddard. Biocontrol Sci. Biol. 6, 539–548. Chen, S.Y., Dickson, D.W., 1996. Fungal penetration of the cystwall of Heterodera glycines. Phytopathology 86, 319–327. Chen, S.Y., Liu, X.Z., 2005. Control of the soybean cyst nematode by the fungi Hirsutella rhossiliensis and Hirsutella minnesotensis in greenhouse studies. Biol. Control 32, 208–219. Chet, I., Inbar, J., Hadar, Y., 1997. Fungal antagonists and mycoparasitism. In: Wicklow, D.T., Soderstrom, B. (Eds.), The Mycota. Volume IV: Environmental and microbial relationships. Springer-Verlag, Berlin, Heidelberg, pp. 165–184. Crump, D.H., Sayre, R.M., Young, L.D., 1983. Occurence of nematophagous fungi in cysts. Plant Dis. 67, 63–64. Leij De, F.A.A.M., Kerry, B.R., Dennehy, J.A., 1992. The effect of fungal application rate and nematode density on the effectiveness of Pochonia chlamydosporia as a biological control agent for Meloidogyne incognita. Nematologica 38, 112–122. Deng, S., Lorito, M., Penttilä, M., Harman, G.E., 2007. Overexpression of an endochitinase gene (ThEn-42) in Trichoderma atroviride for increased production of antifungal enzymes and enhanced antagonist action against pathogenic fungi. Appl. Biochem. Biotechnol. 142, 81–94. Elad, Y., 2000. Trichoderma harzianum T39 preparation for biocontrol of plant diseases control of Botrytis cinerea, Sclerotinia sclerotiorum and Cladosporium fulvum. Biocontrol Sci. Tech. 10, 499–507. El-Alfy, A.T., Schlenk, D., 2002. Effect of 17{beta}-estradiol and testosterone on the expression of flavin-containing monooxygenase and the toxicity of aldicarb to Japanese Medaka, Oryzias latipes. Toxicol. Sci. 68, 381–388. Evans, K., Trudgill, D.L., Webster, J.M., 1993. Plant Parasitic Nematodes in Temperate Agriculture. CAB International, Wallingford, UK.

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