Rhizoctonia solani

Three RNA-seq datasets from the industrial T. versatilis were at our disposal (unpublished data) and were prepared from: 1) growth of the mycelium on MM for 48 h (reference condition); 2) transfer of the water-rinsed mycelium to MM with ball-milled wheat straw 1% (w/v) as carbon source and sampling after 24 h; 3) direct addition of glucose at 1% final concentration to the mycelium exposed to wheat straw, and sampling after 5 h. These RNA-seq data were used for the pre-selection of stable genes (fold change (FC) equal to one, see Additional file 2) after calculating the FC as follow: RPKM (Reads Per Kilobase of exon model per Million mapped reads) value in the sample of interest / RPKM in the reference condition, for each gene. Similarly, FC for candidate reference genes were calculated from RNA-seq data publicly available at the NCBI GEO database [48 (link),49 (link)]. To identify the homologues of T. versatilis selected reference genes in the different fungi, a standard protein BLAST (blastp) using the amino-acid sequence from T. versatilis was performed against protein databases, specifying the organism. Each homologous sequence was then used for a reciprocal BLAST against the T. versatilis database in order to confirm the accuracy of the result. The detailed list of locus tags for each gene in every fungus is available in the Additional file 3. For each GOI in these studies, the ratio between the expression in a condition of interest and the expression in the control condition was calculated. Collected datasets were from Trichoderma reesei ([50 (link)], accession #GSE44648), Aspergillus niger ([51 (link)], #GSE33852), Aspergillus flavus ([52 (link),53 (link)], #GSE40202 and #GSE30031), Aspergillus fumigatus (#GSE30579), Aspergillus oryzae ([54 (link)], #GSE18851), Aspergillus nidulans ([55 (link)], #GSE44100), Blumeria graminis ([11 (link)], #GSE43163), Colletotrichum graminicola ([56 (link)], # GSE34632), Colletotrichum higginsianum ([56 (link)], #GSE33683), Fibroporia radiculosa ([57 (link)], #GSE35333), Magnaporthe oryzae ([58 (link)], #GSE30327), Neurospora crassa ([55 (link)], #GSE44100), ([59 (link)], #GSE35227), ([60 (link)], #GSE36719), Pyronema omphalodes ([61 (link)], #GSE41631), Rhizoctonia solani ([12 (link)], #GSE32577), Sordaria macrospora ([62 (link)], #GSE33668). We also accessed unpublished data from Rhizophagus irregularis ([7 (link)], #SRX375378 at NCBI Short Read Archive) and Leptosphaeria maculans (personal communication from T. Rouxel, INRA-Bioger, Thiverval-Grignon, France).

The water extract from A. saligna flowers was prepared at concentrations of 0%, 1%, 2%, and 3% by dissolving the extract in 10% dimethyl sulfoxide (DMSO). A total of 36 wood samples of Melia azedarach with dimensions of 0.5 cm × 1 cm × 2 cm, air-dried, and autoclaved at 121 °C for 20 min, were used for the antifungal activity test (Figure 5). Three wood samples were used for each concentration. The antifungal activity was evaluated against the linear growths of Fusarium culmorum, Rhizoctonia solani, and Penicillium chrysogenum. The inhibition percentage of mycelial growth was calculated using the following equation: Mycelial growth inhibition (%) = [(A_C − A_T)/A_C] × 100 [77 (link)], where A_C and A_T are average diameters of the fungal colony of the control and treatment, respectively. Wood samples soaked only with 10% DMSO were used as the control.

For each treatment three microcosms were randomly selected to isolate bacterial strains (40 per replicate). Bacterial isolates were randomly selected from the most abundant bacterial colonies (colonies developing from bacterial cells that were present in the most diluted sand suspensions). Bacterial isolates were individually screened for the possession of enzymes that could be involved in destabilization of fungal cell walls namely chitinases, ß-1,3- glucanases and proteases [22 ]. Tests for the production of chitinases and ß-1,3- glucanases were done as described by [17 ], except that the agar for ß-1,3- glucanase detection contained 0.5 g L^-1 laminarin (Sigma,) instead of lichenan. Proteolytic activities of bacterial isolates was tested by the production of clear zones (haloes) on 1/10 strength TSB agar containing 80 ml L^-1 skimmed milk.
The bacterial isolates were also screened for in vitro antagonistic activities against the fungi used in this study as well as against the plant pathogenic fungus Rhizoctonia solani (anastomosis group 2.2IIIB). The in vitro antagonism tests were performed on 1/10 strength TSB agar as described by De Boer et al. [17 ].

Microbial strains were characterized based on morphological [18 ] and biochemical properties including cellulase and catalase production tests [18 ] (Table 1). The plant growth-promoting activities e.g., phosphate solubilization [19 ], indole-3-acetic acid (IAA) by using Salkowski reagent and a few drops of orthophosphoric acid [20 (link)], ammonia production by using Nessler's reagent [21 ], siderophore production by the use of CAS (Chrome Azurol S media) as described by Schwyn and Neilands [22 (link)] were estimated (Table 2). Bio-controlling activities was estimated on dual media plate by inoculation of Fusarium oxysporum and Rhizoctonia solani with bacterial strains [8 (link)] (Fig. 1A and B). Amylase test was done by using preparing 0.1% starch agar media, urease, citrate, and MRVP test was done [18 ].Table 1

Biochemical characterization of isolated bacterial strain.

Table 1

Strains	Biochemical characterization
	Amylase	Catalase	Urease	Citrate test	Methyl red	Voges-Proskauer
Pseudomonas sp.IESDJP-V1	++	+++	–	+	–	–
Pseudomonas sp.IESDJP-V2	++	++	–	++	–	–
Serratia marcescensIESDJP-V3	++	+	+	+	–	+
Bacillus cereusIESDJP-V4	++	++	+	–	–	+
Ochrobactrum sp.IESDJP-V5	+	+	–	–	–	+
Azospirillum brasilensisMTCC-4037	+	+	+	+	+	–
Paenibacillus polymyxaBHUPSB17	+	++	–	+	–	–

*Note: In this table “+++”, “++”, “+” and “-”represent the production ability of microbes in high, moderate, low and absent, respectively. All experiment was conducted with 3 replications setup.

Table 2

Characterization of plant growth promoting biochemical activities of isolated strain.

Table 2

Strains	Phosphate solubilization (μgml−1) at 3days	IAA production (μg ml−1) at 48 h		Siderophore production	Ammonia production	HCN production	Biocontrol activity
		150 μgml−1 tryptophan	300 μgml−1 tryptophan				Fusarium oxysporum	Rhizoctonia solani
Pseudomonas spIESDJP-V1	39.25 ± .66e	30.05 ± .86f	34.86 ± .17e	++	+++	++	+	++
Pseudomonas spIESDJP-V2	33.02 ± .14c	18.27 ± .60b	32.06 ± .05d	+	++	+	+	++
Serratia marcescensIESDJP-V3	33.30 ± .16c	23.70 ± .35d	26.59 ± .07c	+	+	+	–	–
Bacillus cereus IESDJP-V4	37.48 ± .44d	20.08 ± .05c	25.23 ± .09b	+	+	+	+	+
Ochrobactrum spIESDJP-V5	24.76 ± .12b	25.30 ± .87e	55.48 ± .08g	+	++	+	–	–
Azospirillum brasilenseMTCC-4037	19.12 ± .12a	40.59 ± 1.18g	52.08 ± .13f	+	++	–	–	–
Paenibacillus polymyxaBHUPSB17	136.14 ± .10f	12.56 ± .18a	23.11 ± .03a	+	+	+	+	+

Note: The data Values are the mean ± SE, mean values in each column with the same superscript (s) do not differ significantly by Duncan multiple post hoc test (P = 0.05). The sign “+++”, “++”, “+” and “-” represent the production ability of microbes in high, moderate, low and absent, respectively. All experiment was conducted with 3 replications setup.

Fig. 1

Pseudomonas sp. IESDJP-V1 showed inhibition zone against Fusarium oxysporum (A) and Rhizoctonia solani (B) on dual media plate of mixture of 50% nutrient agar and 50% Potato dextrose agar

Fig. 1

Athelia rolfsii (Curzi) Tu & Kimbrough (provided by the Belgian Co-Ordinated Collections of Microorganisms “BCCM”/Agro-Food and Environmental fungi MUCL 051031) was reactivated on V8 juice agar (200 ml of V8 juice; 3 g of CaCO₃; 15 g of agar; 800 ml of distillated water; pH = 7.2). After this step, fungus was cultivated on Oatmeal agar OMA (Per liter: 60 g of oatmeal; 12.5 g of agar, pH = 7.2 ± 0.2) to produce mycelia and sclerotia at 28°C. Inoculated petri dishes were stored at 4°C or collected sclerotia were retained in peptone water (10 g/l of peptone, 5 g/l of NaCl, 1-2 g of tween 80) at the same temperature for the next experiments.
Fusarium oxysporum, Aspergillus niger and Rhizoctonia solani (lab collection) were cultivated on Potato Dextrose Agar PDA (39 g/l, pH = 5.6 ± 0.2; Merck Germany) to produce mycelia. Cultivated fungi were stored at 4°C for subsequent experiments.

Ten µL of GA1 WT or mutants’ strains (OD_600nm = 1) prepared as described in section 2.1 were spotted at 4.5 cm of the pathogen (A. rolfsii) on gelosed MMPRE. Fusarium oxysporum, Aspergillus niger and Rhizoctonia solani antagonism were tested in the same conditions on PDA plates. The plates were sealed with parafilm and incubated at 28°C for 6 days. The inhibition of mycelial growth was evaluated by measuring the distance between bacteria and the advancing front of the fungus. This experiment was performed using 6 independent repetitions. Impact of Bacillus volatiles in antagonism was performed using bipartite petri dishes. In this case, one compartment containing 10 ml of gelosed MMPRE was inoculated with 10 µl of GA1 culture (OD_600nm = 1) prepared as detailed in section 2.1 and sclerotia were deposited at 4.5 cm from bacteria in the other part of the petri dish. The petri dishes were doubled sealed with parafilm and incubated at 28°C for 6 days. Inhibition rate (IR) was calculated with the formula: IR (%) = (S1-S2/S1) x 100 where S1: represents the fungal area growth without bacteria, and S2 represents the fungal area growth in inoculated plates. This experiment was performed using 6 independent repetitions.