Ritu Mawar · Deepesh Sharma · Ladhu Ram

Abstract This study investigates the potential of native biocontrol agents (BCAs) as controls against Ganoderma lucidum causing root rot mortality in Indian mesquite. The disease is prevalent in sandy soils where trees grow under rainfed conditions. In addition, a beetle namely Acanthophorus serraticornis damages the roots, resulting in increasing vulnerability of the host thereby allowing easy of the pathogen. In dual culture tests, Ganoderma infected cowpea root bit experiment and compatibility with insecticides revealed that the three BCAs ( Trichoderma longibrachiatum, T. harzianum, and Aspergillus nidulans) signif icantly inhibited G. lucidum mycelial growth. The highest mycelial growth inhibition (47.6%) was recorded after 96 h followed by 39.8% and 29.3% at 72 and 48 h, respectively, by T. longibrachium. Cell free f iltrates of T. longibrachiatum, T. harzianum, and A. nidulans were superior in inhibiting mycelium growth. A low concentration (3 ml) of T. longibrachiatum was more Effective in inhibiting mycelium growth compared to other BCAs. Both Prosopis julif lora compost and onion residue compost amendments as food substrates favored the growth of these BCAs, which ultimately reduced the viability of Ganoderma- colonized root bits of cowpea. Studies on compatibility between insecticides and BCAs suggests that T. longibrachiatum, harzianum and A. nidulans can be combined with phorate or chloropyriphos (both organophosphates) at variable concentrations if amended together for partially infected trees, or as a prophylactic measure in healthy trees. These studies demonstrate that there is considerable opportunity for using native BCAs against G. lucidum in managing root rot disease.

Keywords Trichoderma longibrachiatum · Trichoderma harzianum · Aspergillus nidulans · Prosopis cineraria ·Onion residues · Prosopis julif lora compost

Introduction

Ganoderma lucidum(Leyss.) Karst. is a destructive root pathogen of various tree and shrub species worldwide,from xerophytic to tropical and temperate regions. It is not freely spread in soils but may live for many years in a saprophytic phase attacking young plantations when their roots cross over infected roots of old diseased plants. In India,decline in plant productivity byGanodermaresults in the death of many species, including cash crops such as coconut,betelnut, tea and tree genera such asAcacia,Albizia,DalbergiaandGrewia(Bilgrami et al 1991; Kumar and Nambiar 1996; Bhaskaran 2000). Under heat and moisture stress conditions in India’s arid regions, many leguminous tree species and shrubs are parasitized by this pathogen (Lodha et al.1986, 1994).

In recent years, these regions have witnessed large-scale mortality due toGanodermaattack onProsopis cineraria(L.) Druce, the Indian mesquite, locally known asKhejri(Lodha and Harsh 2009). This species is an important component of agroforestry systems prevalent in the region, where crops grown in association with this species perform better than those grown without it. Therefore, growers traditionally maintain adequate populations of this species in agricultural f ields for better crop production. Several factors contribute to the weakening of India mesquite leading to infection byGanoderma. The species has a deep root system, drawing moisture from deep strata of the soil. However, the introduction of irrigation or the increase in rainfall results in the development of lateral roots, which draws soil moisture from upper layers of the soil strata (up to 60 cm). In this region,the temperature of the top soil layer often reaches 50-55 °C during summer months (Lodha 1995). At times of concurrent heat and moisture stress, the lateral roots become vulnerable toGanodermaattack; otherwise, it is considered a weak pathogen. The association of an insect,Acanthophorus serraticornisOliver, a longicorn beetle, further aggravates the problem, causing injury leading to the weakening of the tree, making it easy forGanodermainfection (Ahmed et al.2004). Sudden decline in an optimum population ofKhejrion arid and semi-arid agricultural lands has caused considerable concern to farmers of these regions who are dependent on this species for vegetable material, fodder, timber and shelter for their livestock during crop free summers, and particularly as a source of livelihood during periods of drought.Past eff orts made for managing the disease have resulted only in partial control (Ahmed et al. 2004; Lodha and Harsh 2009). Cultural methods of disease control are ineff icient in minimising inoculum load and in reducing disease incidence. Fungicidal controls in the f ield have remained inconclusive (Idris and Ismail 2002), asGanodermahas various resting stages such as melanised mycelium (vegetative fungal spores), basidiospores and pseudosclerotia that are more resistant to fungicides (Susanto et al. 2005). Bivi et al. ( 2010) observed a low incidence of basal stem rot in natural stands in areas where another pathogenic species,G. boninense, was present. This led to the assumption that the disease could likely be kept under control by biological means. Several biological control agents (BCAs), including fungal species ofTrichoderma(Sariah et al. 2005; Susanto et al. 2005),Burkholderia(Sapak et al. 2008),Bacillus(Suryanto et al. 2012) andPseudomonas(Bivi et al. 2010),have been Effective in controllingG. boninensecolonization and infection in nursery and f ield conditions for oil palms and other species. BCAs off er an attractive alternative for the management ofGanoderma-induced diseases without any negative impact on the environment. This is because BCAs colonize the rhizosphere and leave no toxic residues compared to fungicides (Dubey et al. 2007). Amending soils with BCAs supplemented with suitable nutrient sources has been suggested as a simple, practical and long-term management strategy to minimizeGanoderma-induced root rot mortality (Bhadra et al. 2016). Since high soil temperatures are a regular phenomenon during the summer months in this region, eff orts are being made to explore heat tolerant native BCAs. They should be pesticide-resistant or tolerant for inclusion in management programs where insect attacks are also associated. In view of the increasing problem with this root rot fungus in the region, this study was undertaken to investigate the potential of native BCAs forG. lucidumcontrol and their compatibility with insecticides like phorate and chloropyrifos for testing at problematic sites.

Materials and methods

Field surveys were conducted at intervals of 30 km over approximately 1100 km 2 covering 20 villages of two arid districts of Rajasthan state, Jodhpur (26° 28′ N, 73° 02′E) and Sikar (27° 21′ N, 74° 44′ E). The purpose was to assess the incidence of the disease and mortality inKhejriby adopting a 0-4 scale, where 0 was completely healthy and 4 as severelyGanoderma-infected, reaching to death. All categories between 0-4 were diff erent stages of infection as per visual observations and also conf irmed by canopy cover recorded by lux meter (l mm -2 ).

Laboratory experiments

Laboratory experiments were conducted at the Central Arid Zone Research Institute in Jodhpur. The soil was a loamy sand, 85% sand, 8.9% clay, 5.5% silt, and 0.03% nitrogen,0.3% organic carbon, 7.0 μg g −1 Olsen P and pH 8.1. Electric conductivity was 0.88 desi seman (dsm −1 ) (soil: water ratio 1:2.5), bulk density 1.56 g cm −3 and 10.4% (w/w) moisture holding capacity (MHC).

Pathogens

Basidiocarps and infected roots were collected fromKhejritrees, washed with sterile water, and the infected roots cut into 0.5-1 cm pieces, surface sterilized (0.1% mercuric chloride) 30 s, and rewashed in sterile water. Fragments were incubated in sterilized bags with wet cotton at room temperature for 8-10 days. When mycelial growth was observed,pieces were transferred on to potato dextrose agar (PDA)medium. The inoculated Petri dishes were incubated at 28°C for 3-5 days before sub-culturing onto PDA slants. Pure cultures of the fungus were obtained by hyphal tip transfer under aseptic conditions. Ten Petri dishes of PDA were prepared by inoculating each with a 5-mm disc of actively growing mycelium and kept at 28 ± 1 °C until further use.

Biocontrol agents

Three microorganisms were isolated from the rhizospheric soil of healthy and infectedKhejritrees from different problematic sites in the region. These were purif ied by serial dilution followed by single spore isolation on PDA Petri dishes, and then transferred to PDA slants.

Dual culture test

The effi cacy of potential microorganisms as antagonists was evaluated for radial growth inhibition on PDA by dual culture technique againstGanodermaisolate fromKhejri. Six Petri dishes (90 mm) were inoculated with mycelial discs of fungal isolates on one side and aGanodermadisc on the other. The colony diameter of the pathogen in control dishes was also recorded. The per cent inhibition of growth of the pathogen was calculated using a standard formula (Vincent 1927). All three microorganisms isolated during the survey of the problematic sites were identif ied at the culture collection laboratory of the Indian Agricultural Research Institute,New Delhi asTrichoderma longibrachiatum, T. harzianumandAspergilus nidulans.

Interaction of Ganoderma with T. longibrachiatum

Interaction between the most Effective antagonistic fungi,T.longibrachiatum, and the pathogen were assayed. A sterile glass slide was placed in the Petri dish and a small amount of melted PDA spread over the slide as a thin f ilm. Five mm discs of 7-day old colonies cut from the margin of the pathogen andT. longibrachiatumwere placed on opposite sides of the slide 30 mm apart on the PDA surface. A 2-ml sterilized distilled water (SDW) was spread onto the plate to prevent drying and then incubated at 28 ± 2 °C for a week.After the incubation period, the point of contact between Trichoderma-pathogen hyphae was stained with lactophenol in cotton blue and observed under a light microscope (40 ×)for the presence of mycelial penetration and cell wall disintegration (Dennis and Webster 1971).

Effect of cell-free f iltrate

The bio-effi cacy of cell-free f iltrates of the BCAs was evaluated in vitro by the standard poisoned food technique againstG. lucidumto ascertain the release of any metabolites by BCAs inhibitingGanodermain culture. Conical Erlenmeyer 500 ml f lasks containing 250 ml of sterilized PDB medium were inoculated separately with 5-mm diameter mycelial discs cut from margin of a 7-day-old culture ofT. harzianum,T. longibrachiatumandA. nidulans.The f lasks were incubated at 25 ± 1 °C for 21 days. After incubation, the broth culture was mixed well by hand blender and fungal mats harvested on Whatman no 1 f ilter paper and a Sartorius Minisart Syringe f ilter was used for spore-free f iltrates for inoculation. The cell free culture f iltrates (3, 5, 7 and 10 ml)were added to the conical f lasks containing 30 ml of PDB and mixed well; 5-mm discs of 7-day-old culture ofG. lucidumwere then inoculated in each f lask. Four f lasks of each antagonist were used as replications for each concentration of f iltrate. OnlyG. luciduminoculated f lasks served as the control. The inoculated f lasks were incubated at 28 ± 2 °C and after seven days, harvested mycelium in were dried at 60 °C for 48 h and weighed.

Optical density (OD) determination

The three BCAs were maintained on PDA plates. Test cultures were prepared in 250 ml conical f lasks containing 50 ml medium (Banerjee et al. 1993). Flasks cooled to ambient temperature were inoculated from PDA slants with BCAs andGanodermaand incubated on a rotary shaker (35 °C,220 rpm, for 2 days). Three f lasks served as three replications. The biomass produced was aseptically homogenized in a hand blender. The OD of serially diluted, homogenized broth was measured (450 nm) spectrophotometrically after 24, 48, 72, and 96 h against a blank of the uninoculated sterile medium on the same wave length. Homogenized broth was dried at 80 °C for 48 h for weight measurements.The effi cacy of the culture BCAs f iltrates onGanodermaat diff erent time intervals was tested by a spectrophotometer(UV-Vis/NIR).

Preparation of composts

Compost was prepared in a 3 × 2.5 m pit, adopting the Indore method for composting (Howard and Ward 1931). Twigs ofProsopis julif lora(Swartz) DC were collected, crushed into 1.0-1.5 cm pieces and placed separately in four layers in the pit. Each 30-cm layer of residue was covered with a 10-cm thick layer of cow dung. The pit was f inally covered with a 5 cm layer of weeds to reduce moisture loss. Approximately 60% moisture was maintained by the addition of 10-15 l of water at intervals of 7-10 days. Two turnings were given every two months, and the mature compost was ready in 5-6 months.

Evaluation of residue-based composts and residue on growth of BCAs

Virulent strains ofT. longibrachiatum,T. harzianumandA.nidulanswere separately multiplied on potato dextrose broth(PDB) for seven days. Dried root pieces of cowpea (Vigna unguiculataL. Walp.) were cut into 1-1.5 cm lengths and sterilized in PDB in 250 ml Erlenmeyer f lasks, and then inoculated with 5-mm discs of actively growingG. lucidumculture on PDA. Root pieces were allowed to be colonized byGanodermaover 15 days. The experiment consisted of f ive treatments: T1: sterile soil +T. harzianum,T. longibrachiatumandA. nidulans+ SDW; T2: T1 + OR; T3:T1 + OR + PJC; T4: T1 + PJC; and, T5: sterile soil + SDW.Five lots of sterile soil, each 300 g, were separately amended with 3% compost or residue, 6-ml BCAs and a combination of both, and then inoculated with approximately 80-90Ganoderma-colonized root pieces, mixed thoroughly and placed in 12-15 pinhole-punctured polyethylene bags for proper aeration. Non-amended lots served as the control.Three replicates were maintained for each treatment. Soil moisture was maintained at 70% of moisture holding capacity (MHC) at regular intervals throughout the experiment for replenishing water loss. Approximately 15-20 colonized root pieces were retrieved at intervals of 20, 40, and 60 days.After surface sterilization with 1% sodium hypochloride,they were placed on PDA Petri dishes to enumerate viable infection ofGanoderma, and compared with non-amended controls as well as with colonized pieces from PDB.

Compatibility of insecticides with BCAs

Compatibility of BCAs with diff erent concentrations of phorate and chloropyrifos under wet and dry soil conditions was ascertained in a laboratory experiment. BCAs were multiplied in PDB for 10 days and fungal mats harvested on f ilter paper and blended for 30 s in 500 ml of sterile water. This suspension was mixed with 7.2 kg f ield soil and allowed to stabilize for seven days. Three 2 g samples were taken from this soil and initial populations ofTrichodermaandA. nidulanswere estimated as described in the section ‘Biological assays’. The soil was divided into two lots of 3.6 kg. Of this,1.8 kg was moistened with sterile water to maintain MHC of the soil. Three sub samples of 300 g were amended with 500, 1000 and 2000 ppm concentrations of phorate or chloropyrifos and again divided into 100 g each, which served as three replications and one lot of 300 g divided into three samples remained as onlyT. harzianum,T longibrachiatumandA. nidulansamended soil, which served as the control.In the same manner, another lot (1.8 kg) with the same insecticide concentrations was prepared and marked as ‘dry’. In another lot (3.6 kg), insecticides at the same concentrations were amended on the start day but BCAs were amended after seven days. These lots were kept in 12-15 pinholepunctured polythene bags and then incubated at 28 ± 2 °C.After thoroughly shaking, a 5 g sample was retrieved from each bag 5 h after the start of the experiment, and after 30 days to estimate populations of both theTrichodermaspecies andA. nidulanson their respective media.

Biological assays

All the media used were from HIMEDIA (Himedia Laboratories Pvt. Ltd. Mumbai, India). The population ofTrichodermaon a selective media was determined (Elad et al.1981). The population ofA. nidulanswas determined on Martin’s Rose Bengal agar medium by the serial dilution technique. Six Petri dishes of each medium were used for enumeration of each category from one sample. The means of six Petri dishes were considered one estimation per replicate of each treatment.

Statistical analysis

All the data obtained from the diff erent experiments were subjected to analysis of variance (ANOVA) and the treatment means were compared by LSD (P< 0.05). Values on compatibility of BCAs and insecticides were subjected to analysis in three factor-factorial design to work out interactions between concentrations, moisture levels and concentration × time. Correlation coeffi cients between dry mycelial weight and OD were determined and regression equations were constructed for OD with homogenized mycelial growth weight ofGanoderma(Snedecor and Cochran 1967).

Results

Fig. 1 Disease incidence of Ganoderma on Prosopis cineraria at different survey sites

During the survey, 10%Ganoderma-induced mortality,and 10-50% disease incidence recorded forP. cinerariatrees in diff erent arid and semi-arid f ields (Fig. 1). Disease incidence was recorded by categorizing and scoring the diseased individuals using a standard 0-4 scale. Mortality in rain-fed areas was 10-15%, while in irrigated areas the extent of mortality was 1-5% with most of the dried and diseased trees removed by the farmers. However, proper care in removing diseased stubble was not carried out.Canopy diameter was recorded with a Lux meter (l mm −2 ).Trees with high canopy diameters were healthier than ones with less canopy diameter. Among 114 trees selected for recording disease incidence, 30.7% were classif ied in disease categories 1 and 3, while 33.3% were in category 2.The balance of 5.3% were in category 4 and considered dead or dying. Trees classif ied in the 0 category were not considered. Among the locations, most of the trees were in 2 category and onwards at Neem ka thana (Sikar district).Diff erent types of fruiting bodies ofGanodermawith variations in morphology were collected. Infected trees were selected at the problematic sites and marked for future treatments in farmers’ f ields.

Fig. 2 Mycelial growth of Ganoderma with T. longibrachiatum (Tl),T. harzianum (Th) and A. nidulans (An) from 24 to 96 h in dual culture test

Dual culture tests

Percent inhibition inG. lucidumgrowth ranged from 80 to 85%, the maximum beingT. longibrachiatum. In dual culture tests, a 49.7% reduction in mycelial growth ofGanodermain the presence ofT. longibrachiatumwas recorded, compared to mycelial growth of pathogen-inoculated Petri dishes in the control (68.7 mm). In the presence ofT. harzianum,a 46.4% reduction in growth ofGanodermawas recorded,while withA. nidulans, this reduction was 43.9% (Fig. 2).Therefore, among the three BCAs, maximum Effectiveness was expressed byT. longibrachiatum. When mycelium of the pathogen and the biological control agents came in direct contact after 48 h of incubation, growth ofGanodermawas checked and the BCA mycelial growth overgrew, leading to hyperparasitism on the mycelium of the pathogen. BCAs coiled around the hyphae ofGanoderma, both sparsely and intensely, followed by penetration of BCAs into the hyphae.This process was more conspicuous on the reverse side of the Petri dishes, and was more prominent after 96 h where whitish growth ofGanodermamycelium was less visible.

Interaction of Ganoderma with T. longibrachiatum

Under microscopic examination, whitish mycelium of Ganoderma was visible only during the initial stage, but then a dense hyphal growth overgrew and completely hyper parasitized the mycelial mat of the test pathogen (Fig. 3).Hyphal coiling hooks, pincer-shaped structures, short contact branches and hyphal depressions were recorded. Further,hyphae of Ganoderma were degraded byT. longibrachiumhyphae.

Effect of cell-free f iltrate

In the liquid culture test, cell-free f iltrates ofT. longibrachiatum, T. harzianumandA. nidulanswere signif icantly superior in inhibiting the mycelium growth ofG. lucidumcompared to the control (Fig. 4). Even a low 3-ml concentration ofT. longibrachiatumwas more Effective in inhibitingGanodermamycelium growth compared to other BCAs.The reduction increased with increasing BCA concentrations. Maximum mycelium growth inhibition (82.6%) was recorded after f ive days of inoculation in a 7-ml culture f iltrate ofT. longibrachiatumcompared toA. nidulans(75.0%)andT. harzianum(60.5%).

Fig. 3 Hyper parasitism of T.longibrachiatum (Tl) against G.lucidum after 24 h of inoculation

Fig. 4 Mycelial growth of Ganoderma in presence of cell free f iltrate(3-10 ml) of T. longibrachiatum (Tl), T. harzianum (Th) and A. nidulans (An)

OD (optical density) determination

Results on the Effect on optical density on 450 nm revealed that, with 3, 5, 7 and 10 ml f iltrates ofT. longibrachiatum,T. harzianumandA. nidulans,the opacity of the solution was negatively correlated (r = − 0.71 to − 0.99) to the inhibited growth ofGanoderma. Data analysis showed that the highest reduction in hyphal growth was with 10 ml f iltrate concentration. Increase in the percent reduction showed linear growth, suggesting continuous increase in the effi cacy of the f iltrate. As the experiment was performed on time and concentration parameters, all the regression equations accounted for 90 to 97% of the variations in the OD and therefore were attributed only to homogenized mycelium of the diff erent BCAs. To quantify the relative contribution of mycelial weight and OD, the multiple regression equations were constructed as:

whereYandxare estimated values of OD and dry mycelial weight ofGanoderma.

Root piece experiment

All treatment combinations showed signif icant reduction in viable propagules ofGanodermain colonized root pieces after 20, 40 and 60 days compared to the controls. However, there were signif icant diff erences in the reduction of viable propagules among all treatments with BCAs. Percent reduction of viable propagules in colonized pieces ranged from 60, 85 and 100% at 20, 40, and 60 days after the inoculation, respectively, compared with the nonamended soil (Fig. 5). Maximum reduction (85.0%) of viable propagules was achieved with the treatment combination(Tl + Th + An + PJC + OR) within 40 days. A sharp upsurge in the population of BCAs was recorded when amended with PJC (Prosopis julif loracompost) and OR (onion residue) combined or alone. However, the maximum increase over the initial population (4.8 × 10 4 to 12.1 × 10 5 cfu g −1 )of BCAs was in the treatment amended with both PJC and OR compared with 8.6 × 10 5 , 4.5 × 10 5 and 3.6 × 10 5 cfu g −1(colony-forming units per gm) in BCAs amended with PJC or with OR alone and non-amended treatments, respectively.

Compatibility of insecticides with bio agents phorate

T. longibrachiatum

Fig. 5 Reduction in Ganoderma colonized bits in residue and residue based compost and BCA amended soil, T1: T. longibrachiatum (Tl),T. harzianum (Th) and A. nidulans (An), T2: Tl + Th + An + onion residues (OR), T3: Tl + Th + An + OR + P. julif lora compost (PJC),T4: Tl + Th + An + PJC, T5: sterile soil

Signif icant variations in the survival ofT. longibrachiatumpropagules occurred with wet and dry soils, times of incorporation (environments) and with diff erent insecticide concentrations. An initial population of 8.5 × 10 4 colony forming unit (cfu) increased to 1-4.2 × 10 5 cfuof soil whenT. longibrachiatumwas incorporated simultaneously, and to 1.2-1.4 × 10 5 g −1 of soil when the BCA was amended seven days after incorporation of phorate under wet conditions (Table 1). In general, the population ofT. longibrachiatumwas not signif icantly greater than the non-amended control under dry conditions in both environments. Invariably, the survival of the BCA was signif icantly better when simultaneously incorporated with insecticide. This study, therefore, suggest that phorate can be incorporated with theT. longibrachiatumBCA with moisture at 500 ppm concentration.

Trichoderma harzianum

TheT. harzianumpopulation increased signif icantly with all concentrations of phorate in both environments and at both moisture levels compared to the initial level of the population (Table 2). The population of theT. harzianumBCA was signif icantly higher than the non-amended control under wet conditions in both environments. However,the survival ofT. harzianumwas greater in all concentrations of phorate in wet and dry soils under both study environments compared to the initial population. However,viable propagules under dry soil conditions were signif icantly lower than the control except with the 500 ppm concentration when both were simultaneously amended.In general, survival of theT. harzianumBCA was higher when added with phorate, compared to its addition after seven days both in wet and dry soils. However, survival of the BCA decreased at 2000 ppm concentrations of phorate in dry soils compared to lower concentrations. This study,therefore, suggests thatT. harzianumcan be simultaneously incorporated in all the concentrations of phorate under wet conditions.

Table 1 Effect of diff erent concentration of phorate on survival of T.longibrachiatum

Table 2 Effect of diff erent concentration of phorate on survival of T.harzianum (cfu g -1 )

Aspergillus nidulans

A. nidulanspopulations were higher after seven days incorporation of phorate in wet soil compared to its addition with insecticide in dry soil (Table 3). However,A. nidulanspopulations increased signif icantly when both were simultaneously amended and with increased concentration of phorate in dry soil. [However, the population decreased when amended after seven days of phorate incorporation in dry soil.] Now in dry soil also higher populations of theA.nidulansBCA was estimated in the non-amended control compared to diff erent concentrations of insecticide after seven days incorporation. These results suggest that this BCA can be amended along with phorate in dry and wet soils or even after seven days of phorate incorporation under wet soil conditions.

Table 3 Effect of diff erent concentrations of phorate on survival of A. nidulans

Chloropyrifos

Trichoderma longibrachiatum

The population increased signif icantly in the presence of chloropyrifos under both environments and moisture levels (Table 4). An initial population of 4.5 × 10 3 g −1 soil increased to 14.6-17.6 × 10 4 cfu g −1 soil whenT. longibrachiatumwas incorporated simultaneously in wet and dry soils. Survival of these BCAs decreased at 2000 ppm compared to lower concentrations in wet soil. However, with simultaneous incorporation, the population ofT. longibrachiatumremained signif icantly higher at all insecticide concentrations but was signif icantly higher at 500 ppm concentrations in dry soils compared to the control. In general, there was a signif icant decline in the population when amended after seven days of incorporating insecticide, compared to the simultaneous addition of both in wet and dry soil.

T. harzianum

The initial population ofT. harzianum(7.5 × 10 4 cfu g −1 of soil) increased at all chloropyrifos concentrations under both environments and moisture levels (Table 5). However,a signif icantly higher population of the BCA was estimated at the start of incorporation compared to incorporation after seven days. At 500 ppm concentration, the population ofT.harzianumremained equal under wet and dry soil conditions. In general, 500 ppm concentration of chloropyrifos may be more appropriate compared to higher concentrations of the insecticide for simultaneous incorporation of both.

A. nidulans.

A signif icant decline in theA. nidulanspopulation was estimated for all insecticide concentrations under both environments and moisture levels except for the 500 ppmconcentration under dry conditions (Table 6). Only at the 1000 ppm concentration of chloropyrifos did the population ofA. nidulansremain signif icantly higher compared to 500 ppm concentration under wet conditions. However,after seven days, a sudden upsurge in population ofA. nidulansoccurred under dry conditions, which was signif icantly higher than for other concentrations and the control.

Table 4 Effect of diff erent concentrations of chloropyrifos on survival of T. longibrachiatum

Table 5 Effect of diff erent concentrations of chloropyrifos on survival of T. harzianum

Discussion

This study of a series of in vitro and soil experiments demonstrates that there is ample scope for using natural biocontrol agents againstG. lucidumto control root rot mortality inKhejriunder the harsh climate of the Indian arid regions. Of the species ofTrichoderma,T. longibrachiatumshowed greater potential thanT. harzianumin inhibitinggrowth of the pathogen. On the basis of surveys, growers were advised to remove all stubble of dead trees infected byGanoderma, which serve as an inoculum source for healthy trees. Microscopic studies showed thatT. longibrachiatummycoparasitized the mycelium ofGanodermaby attaching longitudinally to the hyphae of the pathogen, coiling and dissolving the pathogen cell walls and membranes by the activity of enzymes. Kishan et al. ( 2017), working with different species ofTrichodermaagainstSclerotia sclerotiumalso showed mycoparasitism of the pathogen due to the activity of enzymes.

Table 6 Effect of diff erent concentration of chloropyrifos on survival of A. nidulans

In our spectrometric determination, optical density was correlated with fresh and dry mycelial weights. Therefore,the optical density of homogenized fungal growth can be a simple, rapid and sensitive indicator of biomass concentration as long as the sample pretreatment by homogenization is carried out under reproducible conditions. Banerjee et al.( 1993) working withNurospora sitophilafound a linear correlation between optical density and dry mycelial weight,and observed that optical density increased with increasing homogenization, reducing turbidity by calculating the size of the fungal hyphae. This study demonstrates that the spectrophotometric determination technique is comparable to that of direct recording of dry weight.

Both theProsopis julif loracompost (PJC) and onion residue (OR) amendments as food substrates favored the growth of these biocontrol agents, which ultimately reduced the viability ofGanoderma-colonized cowpea root pieces. The use ofP. julif loraas decoction has been highly Effective in promoting the growth ofT. harzianum(Lodha et al. 1999).The reason for the increased BCA growth was attributed to the presence of high amounts of sugar and small amounts of tricontanol, a growth hormone in the pods ofProsopisspp.(Khan et al. 1992). In earlier studies, the amendment of soil withP. julif loraresidue-based compost increased concentration of micronutrients, soil moisture, microbial antagonism,and signif icantly reducedMacrophomina phaseolina(Tassi)Goid induced charcoal rot mortality in legumes (Bareja et al.2013). Similarly, residues of onion were Effective in managingGanoderma-induced mortality in Indian mesquite due the promotion of a native strain ofA. terreus, a BCA against this pathogen, and to the presence of certain anti-fungal compounds (Lodha and Harsh 2009). The Effectiveness of onion or its extract in inhibiting fungal growth is well-documented (Nwinuka et al. 2006). In another study, aqueous and ethanol extracts ofAllium thunbergiG. Don (Japanese onion) completely inhibited spore germination ofFusarium proliferatumisolate F801 andColletotrichum higginsianumisolate PA-01 (Hsieh et al. 2005). One antifungal peptide,allicepin, has been isolated from onion and inhibited mycelial growth of several fungal species (Wang and Ng 2004).

Chloropyrifos is a broad spectrum, non-systemic contact insecticide used against a wide variety of insects and acts only when the insect entered in plant tissues then only it can act on nervous system of insect by inhibiting the acetylcholinesterase enzyme. The root treatment of infected trees,using a combination of chloropyrifos (0.02%) + carbendazim(0.1%) is most Effective in killing the larvae ofAcanthphorus serraticornisin the same region (Ahmed et al. 2004).Phorate is an organophosphorus systemic insecticide to control numerous insects (Pohanish 2015). It checkedAcanthophorusinfestation inKhejri(Lodha and Harsh 2009).Our studies on compatibility between biocontrol agents and insecticides suggests that they can be combined with phorate in all concentrations if amended together in partially infected trees or as a prophylactic measure for healthy trees. Similarly, in case of chloropyrifos, the population ofT. longibrachiatumdeclined at higher concentration under both wet and dry soil conditions and could survive up to 500-1000 ppm concentration of insecticide. Jebakumar et al.( 2000) also observed that there was no signif icant diff erences in the viable colonies ofT. harzianumin soils,but rather incorporation of chloropyrifos increased counts of BCA. Utilization of phosphorus from the insecticide was attributed as a possible reason for increased counts of BCA.Kumar et al. ( 2009) observed that chloropyrifos and phorate can be used safely in combination withT. viride. Similarly,Bhai and Thomas ( 2010) reported that carbofuran, copper oxychloride and phorate were highly compatible toT. harzianum. Singh et al. ( 2014) also reported the compatibility of chloropyrifos with many otherTrichodermaspp. but 18.3%inhibition was observed withT. longibrachiatum. The differences with our results may be attributed to experimental variation, as in our experiments soil was used as a testing medium, whereas other edaphic factors such as the content of sand, silt and clay may also play a vital role. Vinale et al.( 2008) reported that the isolates ofTrichodermavaried in their inherent properties of biocontrol, including compatibility with chemical pesticides.

Susanto et al. ( 2005) isolated a number of BCAs againstG. boninensecausal agent of basal stem rot in oil palms from Indonesia and found thatT. harzianumandGliocladium virideare more superior compared to use ofBacillussp. However, in the present study,T. longibrachiatumproved more promising thanT. harzianum,indicating that environmental and edaphic factors may favor enhanced potentiality of some species. This was also evident in our studies of cell-free cultural f iltrates, where maximum inhibition ofGanodermawas observed with ofT. longibrachiatumf iltrate compared to other BCAs, even at low concentrations, indicating a possible release of specif ic metabolites. Therefore,increased survival and multiplication ofT. longibrachiatumwith increasing concentrations and a proportionate reduction inGanodermapropagules recorded in this study are positive signs of the suitability of this BCA againstGanodermain arid soils. The biocontrol potential ofT. longibrachiatumhas remained as a subject of investigation in several countries against fungal pathogens, nematodes, and insects. This biocontrol agent has been found to be an Effective antagonist against root knot nematodeMeloidogyne incognita,and the mechanism was attributed to parasitism and inhibition(Zhang et al. 2015). It has also been reported to be Effective againstHeterodera avenaewhich causes molya disease of wheat and barley, as the activity of b-1,3-glucanase, total f lavonoids and lignin content increased in wheat roots (Zhang et al. 2017). This cyst nematode causes heavy losses in certain districts of the Indian arid regions where wheat is grown in irrigated pockets during the winter season. Therefore, the use ofT. longibrachiatumin these districts should have an additional advantage of controlling serious nematode problems in wheat. Anwar et al. ( 2016) reported on the Effectiveness ofT. longibrachiatumover the nymphal stage of the silverleaf whitef ly,Bemisia tabaci(Gennadius) from Pakistan.T. longibrachiatum,when delivered as a wheat bran,saw dust preparation, provided signif icant protection againstRhizoctonia solani(Kuhn)-inciting root and stem rot of groundnut (Sreenivasaprasad and Sreenivasaprasad 2008).This study conclusively demonstrates that the three BCAs possess potential for managing theGanodermapathogen,as they have also shown good compatibility with both the insecticides. Their use with compost prepared from locally available on farm residues holds promise for better Effectiveness in controlling the incidence of root rot on this valuable species.Prosopis julif lorais abundant in these areas, which often creates a problem for the successful cultivation of agricultural crops.

Acknowledgements Authors are grateful to SERB-DST, New Delhi,Government of India, for providing f inancial support under Core Research Grant.

Compliance with ethical standards

Conf lict of interestThe authors declare that they have no conf lict of interest.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affi liations.