Internal transcribed spacers ( ITS ) based identification of Trichoderma isolates and biocontrol activity against Macrophomina phaseolina , Aspergillus niger and Meloidogyne incognita

Ten Trichoderma isolates were isolated from different locations in Egypt. Amplification and sequencing of internal transcribed spacers (ITS) was employed to identify Trichoderma isolates that exhibited from 99 to 100% identity with three species of Trichoderma: Trichoderma harzianum, Trichoderma asperellum and Trichoderma longibrachiatum. The biocontrol activity of Trichoderma isolates against Macrophomina phaseolina, Aspergillus niger and Meloidogyne incognita was tested in vitro and under greenhouse conditions. The results show that the isolate Th2 (T. harzianum) gave the best antagonism against M. phaseolina and A. niger with inhibition rates of 72.85 and 64.28%, respectively. Moreover, the isolate Ta1 (T. asperellum) was the best efficient isolate in reduction of each second stage juveniles (J2), number of galls, egg masses and females per root system with 90.33, 90.59, 90.06 and 85.50%, respectively. Treatment with Trichoderma isolates improved tomato growth parameters (root length, plant height, roots and shoots fresh weight and shoots dry weight).


INTRODUCTION
The chemical pesticides are the first choice for farmers to control plant pathogens in order to obtain abundant crop yield (Junaid et al., 2013).It was estimated that 12% of crop loss is due to plant pathogens (Sharma et al., 2012).The long term uses of pesticides can cause severe environmental and health problems in addition to their expensive costs for developing countries.Trichoderma is a famous saprophytic fungus that can be isolated from any soil and can be used as efficient biocontrol agent due to its high ability to reproduce and colonize (Pandya et al., 2011;Pal and Gardener, 2006;El-Hassan et al., 2013;Rao et al., 2015).Trichoderma fight against many fungal phytopathogens such as Phythium, Phytophthora, Macrophomina, Aspergillus, Rhizoctonia and Fusarium *Corresponding author.E-mail: k_dein2001@yahoo.com.Tel: 00966542164920.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Trichoderma longibrachiatum using many biocontrol weapons as mycoparasitism, production of antibiotics and competition of pathogens for food and space (Benítez et al., 2004;Rayatpanah et al., 2011;Krishnamurthy and Shashikala, 2006).Recently, the global attention rely on Trichoderma as safe alternative of pesticides and this led to increase in the number of Trichoderma biocontrol products (Woo et al., 2014).
Macrophomina phaseolina and Aspergillus niger are considered destructive pathogens of many important and economically crops causing huge loses of each quantity and quality of crop yield (Khaledi and Taheri, 2016;Njoki et al., 2017;Bhale et al., 2013).M. phaseolina is a dangerous fungus that can infect many of important crops as corn, cotton, sesame, soy bean and sunflower causing diseases such as soy bean charcoal rot (Khaledi and Taheri, 2016;Aly et al., 2007).In addition, A. niger is a common saprophytic fungus that can cause dangerous diseases as black-rot and collar rot in crops like onion and peanut leading to yield loss from 5 to 40% (Khokhar et al., 2012;Gajera et al., 2011;Bhale et al., 2013).Moreover, root knot nematode (Meloidogyne incognita) is one of virulent parasites that attack many crops and can lead to destructive damages in crop yield range from 40 to 50% (Bakr et al., 2011;Katooli et al., 2010;Sikora and Fernandez, 2005;Karssen and Moens, 2006;Maqbool and Kerry, 1997).The previous studies indicated a vital role for Trichoderma species in biocontrol of M. phaseolina, Aspergillus species and M. incognita (Al-Hazmi and Javeed, 2016;Khaledi and Taheri, 2016;Mendoza et al., 2015;Shoaib et al., 2018;Gajera et al., 2011;Sharon et al., 2011).As the cell wall of nematodes and pathogenic fungi is mainly composed of chitin, Trichoderma have chitinase enzymes able to degrade the cell wall of these pathogens (Loc et al., 2011;Haran et al., 1995;Ike et al., 2006).Identification of Trichoderma spp. to be applied in the field of biological control is an important issue.It was reported that identification of Trichoderma based on morphological characters can give misleading results (Fahmi et al., 2016).Recently, molecular identification based on internal transcribed spacers (ITS) amplification and sequencing is common and highly trusted (Savitha and Sriam, 2015;Fahmi et al., 2016;Oskiera et al., 2015;Jiang et al., 2016).
In this study, 10 isolates of Trichoderma spp.were isolated from soil rhizosphere of different locations in Egypt, characterized on molecular level and screened for their antagonistic ability against M. phaseolina, A. niger and root knot nematode.

Isolation of Trichoderma isolates
Ten (10) isolates of Trichoderma spp.were isolated from soil rhizosphere of different sites in Egypt (Table 1) cultivated with different crops as described by Fahmi et al. (2016).Several soil samples were obtained from a 15 cm depth, placed in sterile bags and transferred to the laboratory for isolation process.Serial dilutions were made and 250 μL of 10 3 dilution was distributed onto petri dishes containing potato dextrose agar medium.The plates were then incubated at 28°C for one week for isolation of single colonies.

Soil borne pathogens
Isolates of M. phaseolina and A. niger were kindly provided by Faculty of Science, Zagazig University, Egypt.

DNA extraction from Trichoderma isolates
DNA isolation of Trichoderma was performed as described by Al-Samarrai and Schmid (2000).The clear sharp bands were indicator for the quality of DNA.

Molecular identification of Trichoderma isolates
Polymerase chain reaction (PCR) was utilized to amplify the internal transcribed spacer regions of Trichoderma using ITS1 (5' -TCC GTA GGT GAA CCT GCG G -3') and ITS4 (5' -TCC TCC GCT TAT TGA TAT GC -3') primers.PCR conditions were performed as described by Loc et al. (2011).PCR products were first purified using QIAquick PCR Purification Kit (QIAGEN Cat. No. 28104).Sequencing was performed using Big Dye Terminator v3.1 Cycle Sequencing Kit in a total volume of 20 μL using 3500 Genetic Analyzer, Applied Biosystems (Daejeon, Korea).

Antagonism of Trichoderma isolates
The antagonism of Trichoderma isolates against M. phaseolina and A. niger was evaluated in dual culture plate technique by measuring the radial growth inhibition of pathogen(s) as described by Abdellateif et al. (2017).

Multiplication of M. incognita
Root-knot nematode, M. incognita was identified by observation of perineal patterns according to Hartman and Sasser (1985).Multiplication was done under greenhouse conditions at the experimental greenhouse, Department of Agricultural Botany, Faculty of Agriculture, Menoufia University, Egypt.Susceptible tomato seedlings Cv.GS (Solanum lycopersicum Mill) was transplanted in plastic pots 15 cm in diameter filled with sterilized clays and soil (1:2 v/v).Seedlings were then inoculated by one egg mass of M. incognita.Agricultural operations were carried as needed.

Preparation of M. incognita inoculum
Two months heavily galled tomato roots previously infested with M. incognita were used to prepare nematode inoculum.Massive infested roots removed from the pots and gently washed using tap water to separate the adherent soil particles.Then roots were cut into small pieces and macerated for two periods of 10 s each at high speed by using a blender.The macerated root solution then placed in a jar containing 0.5% of sodium hypochlorite (NaOCl) as described by Hussey and Barker (1973).The Jar was vigorously shaken for 3 min to release the eggs from the egg matrix with the help of NaOCl.The resulting solution then poured through serial sieves to separate the root tissue.Eggs were collected on the last sieve (20 μm) and washed several times with tap water to eliminate NaOCl residuals.The eggs were then transferred to a flask containing tap water.The number of eggs/ ml was estimated by counting 4 samples of 1 ml using a counting dish under a stereomicroscope.

Preparation of Trichoderma inoculum
The Trichoderma isolates were cultured on potato dextrose agar (PDA) and incubated at 26°C for 10 days.The conidia were collected from the surfaces of plates by flooding with sterile distilled water and gently scraping the colony surface with a sterile scrapper (Jansson et al., 1985).

Greenhouse experiment
Greenhouse experiment was carried out to evaluate the effect of 10 Trichoderma isolates on M. incognita.Four weeks old tomato seedlings (Cv.GS) were transplanted into plastic pots (15 cm  diameter) containing sterilized sand-clay soil (2:1 v/v).Each plant is inoculated with 3000 eggs of M. incognita and Trichoderma spore suspension (10 8 g -1 of soil) around the young hairy roots and were mixed well within the pots.Control treatments included untreated seedlings and nematode treated seedlings without Trichoderma.
Treatment with vydate (abroad spectrum nematicide) was used for comparison.
The treatments were replicated three times in a completely randomized block design under greenhouse conditions.Tomato plants were observed, watered and fertilized with a nutrient solution according to Epstein (1972).

Plant growth and nematode parameters
Two months after nematode inoculation, tomato plants were carefully uprooted.The roots were cautiously washed with running tap water.Plant growth parameters as root length (cm), plant height (cm), fresh weight of roots (g), fresh weight of shoots (g) and dry weight of shoots (g) were recorded.Number of galls/root system was counted directly, while number of second stage juveniles (J2S)/250 g soil was evaluated using serial sieves and modified Baermann technique as described by Goody (1957).Egg-masses were stained prior to counting by dipping the infected roots in 0.015% Phloxine-B solution for 20 min as described by Daykin and Hussey (1985).Number of females/root system was determined by cutting the root system to small pieces and submerging the roots in a beaker full of tap water for four days at room temperature until they became soft.Roots pieces were then washed through 500 and 250 μm sieves to separate the females from the root debris and counted under a stereomicroscope.

Data and cluster analysis
BLASTn was used to compare Trichoderma sequencing results with known sequences on NCBI site (http://www.ncbi.nlm.nih.gov/).The alignment and phylogenetic analyses were conducted using MEGA version 6 (Tamura et al., 2013).The obtained data of antagonism was analyzed using costat 6.3 version program.Analysis of variance and comparison of means were done at the 5% level of significance according to the Duncan's multiple range test (1955).

Isolation and molecular identification of Trichoderma isolates
Ten isolates of Trichoderma were isolated from rhizosphere soil samples collected from different sites in Egypt (Table 1).PCR based on ITS primers was used to amplify ITS region and gave one band about 600 bp.The PCR products were sequenced and the sequencing data was entered on NCBI site to search BLAST and compare these data with published ITS data.The amplified ITS regions of Trichoderma isolates were exhibited from 99 to 100% identity with three species of Trichoderma; five isolates were found to belong to Trichoderma harzianum (Th1, Th2, Th3 Th4 and Th5), four isolates classified as Trichoderma asperellum (Ta1, Ta2, Ta3 and Ta4), while one isolate (Tl) was identified as Trichoderma longibrachiatum (Table 1).
The phylogeny analysis supported the ITS identification  and divided the isolates of Trichoderma into three clusters, the first cluster included the isolates of T. harzianum (Th1, Th2, Th3, Th4 and Th5).The second cluster included the isolates of T. asperellum (Ta1, Ta2, Ta3 and Ta4); finally, the third cluster contained the isolate Tl of T. longibrachiatum (Figure 1).These results confirm the efficiency of barcode DNA and are compatible with previous studies that employed ITS to identify Trichoderma spp.(Wu et al., 2017;Fahmi et al., 2016;Savitha and Sriram, 2015;Oskiera et al., 2015).

Chitinase gene of Trichoderma
However, chitin is key component of pathogen cell walls, Trichoderma secrete highly active chitinases that can destroy these cell walls and feed on them (Seidl-Seiboth et al., 2014;Hassan et al., 2015;Prasetyawan et al., 2018).Chitinase 42 is one of Trichoderma endochitinases that can degrade the β-1, 4-glycosidic bonds between the N-acetyl glucosamine residues of chitin (Hassan et al., 2015).In this study, PCR based specific primers (CHI-F and CHI-R) was utilized to amplify and detect the chitinase 42 gene in Trichoderma isolates as shown in Figure 2. The PCR gave one band about 1500 bp homolog to that obtained by Loc et al. (2011) and confirmed the presence of this gene in all tested isolates.The amplification of chitinase gene confirms that these isolates are Trichoderma since the primers are specific for Trichoderma chitinases.

Evaluation of Trichoderma antagonism against M. phaseolina and A. niger
The antagonism of Trichoderma isolates against M. phaseolina and A. niger was evaluated in vitro by measuring the radial growth inhibition of the two pathogens (Table 2 and Figure 3).
The isolates Th2, Ta2, Th3 and TI were the most efficient isolates in suppression growth of M. phaseolina   with 72.85, 71.43, 67.14 and 65.69%, respectively.In addition, the isolate Th5 gave only 25.71% radial growth inhibition against M. phaseolina.For antagonism of A. niger, the isolates Th5 and Th2 were the most active isolates in suppression growth of A. niger with 71.43 and 64.28% respectively, while the isolate Tl exhibited the least radial growth inhibition against the same pathogen (31.43%).In general, the isolate Th2 of T. harzianum showed good antagonism against M. phaseolina and A. niger with inhibition rates 72.85 and 64.28%, respectively.Previous studies explained that T. harzianum is more vigorous in inhibition growth of pathogenic fungi than the other species of Trichoderma (Abdel-lateif et al., 2017;Shoaib et al., 2018;Al-Hazmi and Javeed, 2016).
Mycoparasitism is one of Trichoderma biocontrol mechanisms that depend on secretion of lytic enzymes like chitinases and cellulases to destroy the cell walls of pathogens (Gajera et al., 2012).It was proved that chitin is major cell wall component of most pathogenic fungi and Trichoderma is excellent producer of chitinases which can degrade the chitin and therefore killing them (Benítez et al., 2004;Agrawal and Kotasthane, 2012;Gajera et al., 2012).
Trichoderma genus is proved to be an excellent control agent in inhibition growth of M. phaseolina, Aspergillus spp.and root knot nematode under in vitro and green house conditions (Al-Hazmi and Javeed, 2016; Khaledi and Taheri, 2016;Khalili et al., 2015;Mendoza et al., 2015;Shoaib et al., 2018;Athira, 2017;Krishnamurthy and Shashikala, 2006).The variability in aggressiveness of the same Trichoderma isolates against M. phaseolina and A. niger may be due to the variation in pathogen cell wall structure and the defense ability of the pathogen.

The effect of Trichoderma isolates on nematode
The efficacy of Trichoderma isolates against M. incognita on tomato was evaluated in greenhouse experiments and the results were compared with those of vydate and  On the contrast, the isolate T. harzianum (Th5) was the least isolate in reduction of each second stage juveniles (J2), number of galls, egg masses and females per root system with 81.72, 75.41, 77.02 and 71.76%, respectively, as compared to vydate.It was shown that the growth of nematode was increased in absence of Trichoderma.These results highlight the significance of Trichoderma as excellent control agent in inhibition growth of nematode (Spiegel et al., 2007;Chen and Dickson, 2004;Sharon et al., 2011).Interestingly, most of the isolates that exhibited high inhibition rate of nematode growth also improved each root length, plant height, roots fresh weight, shoots fresh, and dry weight of tomato plants (Table 4).These results are compatible with other studies showing the ability of Trichoderma in growth inhibition of nematode on various crops and enhancing the growth of host plants (Al-Hazmi and Javeed, 2016;Izuogu and Abiri, 2015;Sharon et al., 2011;Mascarin et al., 2012).
The variability among Trichoderma isolates in their aggressiveness against the tomato nematode may be due to the difference in their genetic structure, origin of isolates, pathogen cell wall structure and quantity of lytic enzymes secreted by Trichoderma (Al-Hazmi and Javeed, 2016;Abdel-lateif et al., 2017).

Conclusion
Identification of Trichoderma based on ITS markers exhibited high efficiency in discrimination among different Trichoderma spp.isolates.Trichoderma isolates showed variability in their aggressiveness against M. phaseolina, A. niger and M. incognita.In general, the isolate Th2 of T. harzianum showed the best antagonism against M. phaseolina and A. niger, while the isolate Ta1 of T. asperellum was the best efficient isolate in reduction of nematode growth.
In general, treatment of tomato with Trichoderma isolates improved their growth parameters (root length, plant height, roots and shoots fresh weight and shoots dry weight) as compared to untreated control.These results confirm the efficacy of Trichoderma as excellent biocontrol agent and also as plant growth promoting.

Figure 2 .
Figure 2. Amplification of chitinase gene in Trichoderma isolates based CHI-F and CHI-R primers, M; 1 kb plus DNA ladder.
the same letter do not differ significantly (P=0.05) according to Duncan's multiple range test".Results are means of 3 replicates for each treatment.

Table 1 .
Isolation sites and identification of Trichoderma isolates based on ITS data

Table 2 .
Radial growth inhibition of Trichoderma isolates against M. phaseolina and A. niger in dual culture plate technique

Table 3 .
The effect of Trichoderma isolates on nematode growth parameters

Table 4 .
The effect of Trichoderma isolates on tomato plant growth parameters.