Impact assessment of Karanja deoiled cake and sundried biogas slurry as a mixed substrate on the nematicidal potential of Purpureocillium lilacinum

2.7.1 Nematode control

Nematode infestation and its control were determined via rating root gall index on the 1–5 scale (Barker, 1985), control efficacy (Sun et al., 2006), eggs/egg mass, egg mass/gram roots, and nematode population/250 cc soil.

The galling index was rated on a scale of 1 = No galling, 2 = trace (<25% of total root system galled), 3 = slight (25–50% of total root system galled), 4 = moderate (51–75% of total root system galled) and 5 = severe (>75% of total root system galled). The following formula calculated control efficacy: - $$\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{c}\mathrm{y}=\left(\mathrm{G}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-G\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)\times \frac{100}{\mathrm{Ga}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{index}\mathrm{control}}.$$

The number of egg masses per gram roots was determined through visual observation. Egg masses were detached gently with a pair of forceps and extracted in NaOCl solution. The egg masses were broken, and eggs were released into the water. The final volume of egg suspension was made up to 100 ml. 10 ml of this was pipette out after thoroughly bubbling and poured in a counting dish to count the number of eggs per egg mass. Modified Cobb's decanting and sieving method, followed by Baermann's funnel technique, was employed to determine the initial and final population of M. incognita in pots (Southey, 1986). Nematode reduction (%) was calculated using the Henderson and Tilton (1955) formula i.e. $$\mathrm{N}\mathrm{e}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{o}\mathrm{d}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\%)=1-\left(\frac{\mathit{IPT}}{\mathit{FPT}}\times \frac{\mathit{IPC}}{\mathit{FPC}}\right)100$$

Where, IPT and FPT = Initial and final population in treatment.

IPC and FPC = Initial and final population in infested control

2.7.2 Plant growth parameters and biomass yield

After seven months, the plant biomass was harvested. Data about plant growth parameters viz. shoot and root length, fresh and dry shoot weight, fresh and dry root weight, and the number of fruits were recorded. Completely expanded leaves were used to determine chlorophyll a, chlorophyll b, and total chlorophyll content, according to Harborne (1998). Likewise, fully expanded leaves were selected for nutrients (N, P, K, and CP) analysis (Tandon, 1995).

3.1.1 Optimization of substrate composition

For the current investigation, we formulated a screening strategy based on in vitro assessments of antagonistic activity apart from the conventional parameter, i.e., spore production, to evaluate substrate compositions for P. lilacinum 6029 to control root-knot nematode. Wheat grains produced the highest spore count (1.5 × 10¹⁰ spores/g) (Table 2). However, among the four combinations of KDC-BGS, the 40/60 ratio produced maximum spores (9.3 × 10⁹ spores/g) significantly (p < 0.05) at par with the 60/40 ratio (8.2 × 10⁹ spores/g) followed by 20/80 and 80/20 (6.4x 10⁸ spores/g and 4.7 × 10⁸ spores/g) respectively. Evidently, the spore count was directly proportionate to the concentration of BGS in the mixed substrate up to a certain extent. It was possibly due to a high percentage of carbon in sundried BGS, which might have helped bring the optimum C/N ratio of the mixed substrate. Carbon sources and C/N ratios influence the viability, endurance, and virulence of fungal spores (Rangel et al., 2004; Shah et al., 2005; Hutwimmer et al., 2008). Besides, the BGS supplementation made the substrates more aerated and porous for microbial activity. P. lilacinum, being saprophytic, in all likelihood, utilized carbon from sundried BGS as an energy source. Hammer et al. (2014) suggested that fungi naturally incline towards minerals and nutrients accessible from organic substrates, thereby corroborating our findings.

Spores from 80/20 and 60/40 ratio of the substrates showed at par virulency inhibiting egg mass hatching up to 90.68 and 89.1% respectively on the seventh day. Spores obtained from wheat grains were least potent as they inhibited only 68.72% of egg mass hatching of M. incognita after a week. Our findings accentuate the significance of biowastes as culture substrates for producing virulent fungal spores for biocontrol applications. Wu et al. (2009) reported varied conidial virulence harvested from different substrate compositions on diamondback moth larvae. Likewise, the conidia from white rice amended with PDB showed more virulence on Dactylaria higginsii than conidia from white rice alone (Wyss et al., 2001). The current study noticeably displays the impact of culture substrates (KDC-BGS) on the virulence of P. lilacinum spores, although a conclusive linkage between nutrition and virulence remains unclear.

The presence of KDC facilitates the production of protease as the activity was directly proportional to the amount of KDC (r² = 0.9761) in KDC-BGS compositions (Fig. 1). Also, the positive correlation between protease activity and egg mass hatching inhibition, implying that that protease enzyme is a major virulent factor in causing infection to the nematode eggs. Maximum protease activity and egg mass hatching inhibition (382.02 U/g & 90.68% inhibition) were seen in 80/20, whereas minimum in 20/80 ratios (236.82 U/g & 71.1% inhibition) among the four combinations of KDC-BGS. Ultrastructural and histochemical studies have inferred the association of hydrolytic enzymes in penetrating the nematode cuticle and eggshell (Chen et al. 2015). We assume that the high nitrogen content in KDC encouraged hydrolytic enzymes in the quick degradation of eggs of M. incognita in the present investigation. Our findings are in corroboration with the study by Khan et al. (2004), who noticed morphological changes in the eggshell and reduction in egg hatching of M. javanica and related all these events with the secretion of serine proteases by P. lilacinus 251.

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Fig. 1

Effect of substrate compositions on protease activity of P. lilacinum 6029. Data are means of four replicates. In each bar, data followed by the same letter are not significantly different at P < 0.05 by DMRT.

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Therefore, based on spore counts and virulence, the 60/40 ratio was chosen as the optimal combination of KDC-BGS for the SSF of P. lilacinum 6029. The fermented product was shade dried, grounded, and mixed with fly-ash (inert carrier) in a 1:1 ratio to develop bio-formulation to control root-knot nematode infection under lab and greenhouse conditions.

3.1.2 Structural characterization of the substrate after fermentation by scanning electron microscopy

Scanning electron microscopy (SEM) of the unfermented and fermented substrate (60/40 ratio) revealed luxuriant fungal growth on KDC-BGS based nutrient medium (Fig. 2 a, b). We found that the dense mycelia of P. lilacinum colonized a larger part of the optimized substrate. The fungal hyphae appeared to grow inside the substrate distorting the surface (Fig. 2c).

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Fig. 2

Scanning electron micrograph of unfermented (a- 10 µm) and P. lilacinum 6029 10-day fermented KDC-BGS (b) at 10 µm (c) 2 µm (closer view).

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3.1.3 Colonization potential of fungal spores

The effect of fungal spores derived from KDC-BGS based formulation and wheat grains (control) on the colonization rate of egg masses is shown in Fig. 3. Colonization of egg masses by P. lilacinum 6029 spores was validated by inspecting the emerging hyphae from the egg mass surface under a stereomicroscope at 40X. On the 1st day, spores from formulation registered a 36.4% colonization on egg mass, but promptly increased to 89.2% on the 3rd day and completely colonized on the 5th day. On the contrary, fungal spores from the traditional substrate, wheat (control), showed a gradual increase in egg mass colonization, recording 100% colonization on the seventh day. An effective parasite should utilize a gelatinous matrix (GM) of nematodes as a source of nutrients and grow on it (Sharon et al., 2007). Since mycelia of P. lilacinum in the present study was detected on the GM, we understand that the fungus withstands the antimicrobial compound in the GM, which is in line with those of Zaki and Bhatti (1990); Eapen et al. (2005) and hence considerably colonized egg masses of nematodes.

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Fig. 3

Percentage of egg masses of root-knot nematodes colonized by P. lilacinum 6029 spores obtained from the formulations (KDC-BGS and wheat grain-based) under in vitro condition. Data are means of four replicates.

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3.1.4 Shelf-life

The shelf life study took place at two different temperature regimes, i.e., 4 °C (low temperature) and 26 °C (room temperature) for six months (Fig. 4). The viability of the sample stored at low temperatures remained constant throughout storage. At 26 °C, the biocontrol agent remained significantly (p < 0.05) viable during the first month of storage. The CFU count gradually declined from the first month onwards, but 53.21% of the initial count of formulation (4.32 × 10⁹ CFU/g) was viable at optimum temperature even after six months of storage. Duan et al. (2008) had noted similar observations and reported that alginate pellets of P. lilacinus were much more stable when stored at low temperatures (4 or −20 °C) than at 25 or 40 °C. The initial increase in the CFU load during the first month and its maintenance till the third month at room temperature suggested sufficient nutrient support from KDC and BGS. After the third month, a decline in CFU indicates a scarcity of nutrients in the formulation. It is noteworthy that no other microorganism other than P. lilacinum 6029 was detected in all Petri plates during CFU count, implying nil contamination in the formulation during storage.

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Fig. 4

Shelf life study of P. lilacinum 6029 formulations stored at 4 and 26 °C for six months. Data are means of four replicates.

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3.1.5 Dose-response relationship

Before the field expression of our bioformulation, a definitive determination of optimum fungal inoculum density was warranted. In this regard, a dose–response relationship was examined (Table 3). We found that the response intensity (egg mass hatching inhibition) was directly proportional to the dose of P. lilacinum spores. Maximum inhibition of egg mass hatching (95.6%) and minimum IT₅₀ (1.41 days) was achieved by 10⁸spores/ml. As the dose was reduced to 10⁷, 10⁶, and 10⁵ spores/ml, percentage inhibition of egg mass hatching also declined to lower values (94.5, 88.7, and 73.7%, respectively). The fungal dose of 10³ spores/ml was found to be the threshold value for P. lilacinum 6029 spores of formulation as a dose lower to it did not affect the egg mass hatching. Fernandez et al. (1989) reported a 97% reduction in the hatching of eggs when the GM was exposed to 10⁹ spores/g of P. lilacinus while only 60% of eggs hatched at 10⁵ spores/g concentration. A dose–response relationship curve suggested that the IC₅₀ value of the formulation was 10⁴ spores/g of the formulation. It is worth mentioning here that since dose efficacies viz. 10⁸ and 10⁷ spores/ ml toward egg mass hatching inhibition was at par (p < 0.05), we picked 10⁷ spores/g for a greenhouse study. A lower dose minimizes the application cost, thereby increasing the net profit of the crop.

3.2.1 Nematode control

The data about the effect of two different substrates based fungal formulation on M. incognita infestation in L. esculentum var. GS-600 is given in Table 4. Greenhouse study showed that T4, i.e., formulation with KDC-BGS as base substrate @10⁷spores/ g of soil significantly (p < 0.05), managed the nematode infestation (62.5%) in tomato plants. Formulations developed on different nutrient sources also adversely affected M. incognita reproduction on tomato crops. The data revealed that the minimum egg masses/g root was recorded in T4 (9.1), followed by T3 (11.8). Although the GM serves as an agent protecting the eggs against microorganisms, we observed a better efficacy of our formulated product in damaging it. Hence, our findings are in contrast with the study proposing that the antimicrobial properties of GM prevent the egg parasitization (Orion and Kritzman, 2001), and in line with Anastasiadis et al. (2008) who demonstrated the potential of P. lilacinus 251 in colonizing the eggs of Meloidogyne spp.

The data indicated that P. lilacinum 6029 greatly reduced the final nematode population per 250 cc of soil, with the lowest population (81) recorded in T4 followed by T3 (110 nematodes per 250 cc soil). The maximum percentage of nematode reduction over IC was observed in T4 (71.8%), implying the collective effect of fungus and its optimized the mixed substrate on nematode control. Our findings confirm the study where soybean cake defatted and coffee husks have acted as a powerful synergist with P. lilacinum against nematodes in tomato (Brand et al., 2010). Here, nitrogen in KDC may enhance protease activity and, subsequently, the fungus’ virulence.

3.2.2 Physiological parameters

The impact assessment of formulation on various plant growth parameters of L. esculentum var. GS-600 viz., root length, shoot length, and respective weight (fresh and dry) are revealed in Table 5. The tallest plant (102.5 cm) was recorded from the T4 treatment, while the shortest appeared in the inoculated control pots (58.62 cm). Employment of formulation improved both fresh and dry shoot weight over the IC. The formulation developed with wheat (T3) and KDC-BGS (T4) as a base substrate resulted in 23.95 and 21.75 cm mean root length. The smallest root length (12.01 cm) was recorded from the inoculated control plants. KDC-BGS based formulation significantly (p < 0.05) suppressed root galls on tomato, thereby showing an 86.11% increase of fresh root weight over IC in T4. Maximum fruit yield (622.32 g/plant) was obtained from T4, followed by T1 (588.67 g/plant) and T3 (465.21 g/plant). The improved plant growth in KDC-BGS based fungal formulation treated soil could be due to the release of nutrients from the organic substrates. The indirect contribution of the formulation to plant growth by limiting the soil's nematode population cannot be ruled out.

3.2.3 Biochemical parameters

Table 6 provides the effect of formulation on the nutrient content in leaves of L. esculentum var. GS-600. A perusal of the data reveals an increase in nutrient uptake by the plants treated with the formulation. N, P, K, and crude protein in T4 were 2.41, 0.65, 1.64, and 15.06%, respectively. The results demonstrated the beneficial use of KDC and BGS in our formulation. Digested slurry, being organic, release fast-acting nutrients upon decomposition. These nutrients easily enter the soil solution, thus becoming straight away available to the plants besides serving as primary nutrients to develop saprophytes (Warman and Termeer, 2005).

Photosynthesis is an important plant growth parameter related to the chlorophyll content (Fig. 5). The total chlorophyll content in leaves markedly increased in the nematode inoculated plants treated with T4 (1.91 mg/g FW) followed by T1 (1.89 mg/g FW) and T3 (1.43 mg/g FW). Inoculated control, T2 had registered least chlorophyll a (0.829 mg/g FW), chlorophyll b (0.305 mg/g FW) and total chlorophyll content (1.134 mg/g FW) in leaves. P. lilacinum does not have residual toxicity and is known to promote plant growth (Nesha and Siddiqui, 2017).

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Fig. 5

Effect on chlorophyll a, chlorophyll b, and total chlorophyll in leaves of tomato plant inoculated with the P. lilacinum based formulations. Data are means of ten replicates. All means are significantly different from each other at P < 0.05 by DMRT.

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