The efficacy of various doses of Purpureocillium lilacinum PLSAU-1 (P. lilacinum PLSAU-1) was evaluated against brinjal root-knot pathogen, Meloidogyne incognita (M. incognita) at different inoculum levels. Experimental pot soil was treated by four doses (0, 1 × 105, 5 × 105, and 1 × 106 CFU/g soil) of P. lilacinum PLSAU-1 before transplanting, and 5 inoculum levels of M. incognita (0, 100, 400, 800, 1600, and 3200 eggs per 100 cm3 soil) were used after 3 days of brinjal transplantation. A significant correlation was observed between doses of P. lilacinum PLSAU-1 and inoculum densities of M. incognita. Among the doses, the rate of P. lilacinum PLSAU-1 of 1 × 106 CFU/g soil enhanced highly plant growth parameters. Inoculation of M. incognita reduced plant growth significantly, and the reduction increased with the increase of inoculum density of M. incognita. The application of P. lilacinum PLSAU-1 at 1 × 106 CFU/g soil reduced a maximum of 72% gall index and 84% egg masses when the crop was challenged by 800 and 400 eggs/100 cm3 soil, respectively. The dose of 1 × 106 CFU/g soil of P. lilacinum PLSAU-1 showed effectiveness to reduce reproduction factor of M. incognita up to 81% when the crop was inoculated by 800 eggs of the pest/100 cm3 soil. The study demonstrated that P. lilacinum PLSAU-1 is an effective bio-agent for controlling of root-knot nematode on brinjal and can be a key component of environment-friendly management approach.
Brinjal (Solanum melongena L.) is a very common, popular, and second most (next to potato) important vegetable crop, in Bangladesh and other parts of the world, in respect of acreage and production. It contains higher calorie, iron, phosphorus, and riboflavin than tomato (Meherunnahar and Paul 2009).
The incidence of insect pests and diseases generally hampered the production of eggplant. In Bangladesh, this crop suffers from 13 different diseases, recorded (Rashid 2000). Among those, root knot has been considered as one of the key constrains in eggplant cultivation. It is caused by Melodoigyne spp., the widely distributed in all eggplant-growing areas of Bangladesh with moderate incidence (27.2%) (Bari 2001) as well as in other countries of the world. Meloidogyne incognita (M. incognita) is the most vicious species among them, which causes severe harms to a quantity of economically significant agriculture and greenhouse crops (Huang et al. 2014).
Infection of roots by nematodes affects uptake of water and nutrients and interferes with the translocation of minerals and photosynthates, which change the shoot to root ratio (Saikia et al. 2013) and expose the plants to other pathogen infection. For example, nematode root infection increases the incidence and severity of wilt and root rot diseases on a variety of crops, which can negatively influence yield (Osman et al. 2018). Controlling of this type of plant-parasitic nematode is essential for effective crop production. Presently, the most proficient chemical nematicides (e.g., methyl bromide) have been constrained due to their hazardous effect on animal and human. Therefore, it has led to an immense interest in biological control in order to achieve an environmentally safe method of reducing nematode damage (Abdelnabby et al. 2011).
Purpureocillium lilacinum is one of the most promising and practicable biocontrol agents for the management of plant parasite nematodes (Yang et al. 2015b). Previous studies indicated that this fungus adapts well in varied climatic conditions and is much effective in controlling root-knot nematodes. This fungus is capable of infecting all life stages of the root-knot nematode. The production of leucinotoxins, chitinases, proteases, and acetic acid by P. lilacinum is associated with the infection process (Yang et al. 2015b). Although P. lilacinum has been reported as a nematode biocontrol agent on plant crops (Usman and Siddiqui 2012 and Yang et al. 2015b), and recently, it also has been reported as a biocontrol agent for controlling Sclerotinia sclerotiorum the rot on oilseed rape (Yang et al. 2015a).
Recently, some works have been done on the effect of fungal strains for the management of root-knot nematode on tomato and cucumber (Kiewnick et al. 2011 and Huang et al. 2014). However, the dose-response relationship and optimum dose for controlling of root knot (M. incognita) on brinjal still remain unidentified.
The main objective of this study was to evaluate the dose effect of P. lilacinum PLSAU-1 on the plant growth parameters, root galling at different inoculum level of nematode, and nematode population and reproduction factor of the root-knot nematode M. incognita at different inoculum levels in brinjal.
Material and methods
Experimental materials and design
The experimental soil was silty loam (13.8% clay, 56.5% silt, and 29.7% sand) with a pH of 5.2 and an organic matter content of 1.5%. Required soil, sand, and decomposed cow-dung were mixed properly in a ratio of 6:2:1. The mixture was autoclaved at 121 °C for 15 min at 15 PSI. Plastic pots of 1000 cm3 were cleaned, washed, dried up properly, and sterilized with 70% ethanol. Then, they were filled with 800 g soil and arranged according to the experimental design. The experiment was laid out in two factors according to a randomized complete block design (RCBD) having eight replications for each treatment.
Inoculums and fungus preparation
Nematode samples (M. incognita) were collected from nematode-infected brinjal roots. Egg masses were picked up and inoculated in young seedlings of brinjal. Sub-culturing was done subsequently by inoculating new brinjal seedling with egg masses. Galled roots of tomato, eggplant, cucumber, and bottle gourd were collected from farmers’ field of different districts (Dinajpur, Narayangonj, Mymensingh, Jessore, and Dhaka) in Bangladesh. The samples were kept in double-layered plastic bags at 48 °C, and fungi were isolated within 1 month. After collection, roots were washed by running tap water and placed under a dissecting microscope. Egg masses were picked up manually, using dissecting forceps, and the roots were dissected beneath the egg masses; females were carefully picked up with a dissecting needle. Eggs were extracted from the egg masses, following the method of Hussey and Barker (1973). Egg masses were treated by 1% NaOCl solution for 1 min to release eggs from the egg masses and disinfect the egg surface, whereas females were treated by 0.5% NaOCl solution for 3 min. Treated eggs and females were rinsed by a sterile distilled water to remove residual NaOCl. One hundred eggs (ca.) were plated and smeared on each potato dextrose agar (PDA) plate; five plates were applied for each sample. Five females were crushed by a sterile forceps and smeared on each PDA plate. All plates were incubated at 25 °C for 5 days. The incubated plates were daily examined under an inverted microscope, and hyphae grown from eggs or females’ fragments were transferred on to PDA plates for purification and identification. The isolates were identified, following the standard keys of Houbraken et al. (2010). This fungus was isolated at the laboratory of Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. The fungus was named as P. lilacinum PLSAU-1 (Aminuzzaman et al. 2018).
Inoculation of P. lilacinum PLSAU-1 and M. incognita
P. lilacinum PLSAU-1 spores were mixed carefully into the soil at (1 × 105, 5 × 105, and 1 × 106 CFU) of P. lilacinum PLSAU-1/g of soil in defined pots with a micropipette. Four-week-old seedlings were uprooted cautiously from the plastic trays and transplanted in the fungal-treated pots. Sufficient water was given just after transplantation. Then, after 3 days, nematode was inoculated at (0, 400, 800, 1600, and 3200 eggs/100 cm3) of pot soil. Four holes (2-cm depth) around the plants were made with the help of metallic rod, and egg suspension was inoculated into the holes by a micropipette. Pot soil contained water only untreated control and nematode only inoculated control. General sanitation was maintained throughout the growing period.
Data recording and analyzing
Plants were harvested after 2 months for collecting data. The shoot length was measured from the base of the plant to the growing point of the youngest leaf. To measure root length, the roots were cleaned gently by water and the length was measured from the growing point of the root to the longest available lateral root apex with measuring tape. After taking root length and fresh weight, nematode infection was determined by rating root galling based on the 0–10 scale (0 = no galls and 10 = dead plant) (Bridge and Page 1980). The number of egg mass/root system was counted by Hartman and Sasser (1985). The roots were soaked in Phloxine-B (2 mg/l) solution for 15 min, and then, the egg masses/roots were counted using a magnifying lens. The extraction of J2 from the soil and root was done, using a Whitehead and Hemming tray method (Whitehead and Hemming 1965). Pot soil was mixed thoroughly, and a 10-g soil from each pot was weighted and placed on a sieve on a bowl filled with water. The upper portion of the sieve was lined by three layers of kitchen tissue paper. After 3 days, the bowl water was collected in a beaker, and then, nematodes were concentrated into 10 ml of water using 500 mesh sieves. The two-milliliter suspension was taken from each sample and placed into a counting dish, and the J2 were counted under a compound microscope. To estimate the eggs per root, roots were gently washed by tap water to remove soil. Then, roots were chopped into 2 cm lengths placed in a 100 ml solution of 0.6% sodium hypochlorite and mixed, using an electric blender. The blended mixture was poured through the sieves from top to bottom (80, 200, and 500 meshes). Eggs were collected from 500 mesh sieve in a beaker and counted using a counting chamber.
For the percentage of egg mass colonization, five egg masses/plant were randomly collected from the eggplant roots, washed by a sterile water, and disinfected by a solution of Clorox rinsed with water and placed on a PDA plate. The number of colonized egg masses was determined after 5 days of incubation at 25 °C. The presence of P. lilacinum PLSAU-1 into egg mass of M. incognita was confirmed by preparation of slides from the culture grown on PDA. Soil colonization by P. lilacinum PLSAU-1 (CFU/g soil) was determined by a soil dilution plate technique. In details, 2 g of soil (wet weight) was transferred to flasks containing 20 ml of sterile demineralized water and shake on a horizontal shaker for 25 min at 170 rpm. After preparation of the serial dilutions, CFU numbers were determined by plating onto OHIO agar (Johnson and Curl 1972). Final nematode population was determined eggs per root plus the 2nd-stage juvenile per pot; reproduction factor = (final nematode population)/(initial nematode population).
The recorded data of various parameters were statistically analyzed using MSTAT-C statistical package program. Differences among treatment means were determined by Duncan’s new multiple range test (DMRT).
Results and discussion
Effect of P. lilacinum PLSAU-1 and M. incognita application rates on plant growth parameters and nematode’s population
Results of the present study indicate significant effects of inoculum level and dose applied on the growth parameters. The treated 1 × 105, 5 × 105, and 1 × 106 CFU/g soil of P. lilacinum PLSAU-1 shoot fresh weight and shoot length were significantly greater than the untreated control (Table 1). The mechanism of improvement of plant growth by the biocontrol fungi is unknown. Our hypothesis is maybe it enhanced nutrient uptake, incorporate mineral nutrition through mineral solubilization, disease suppression, or involved production of phytohormones. Also, it may be caused when this bio-agent parasitizes other pathogenic fungi and competes with harmful plant microorganisms (Contreras-Cornejo et al. 2009).
For the inoculum level, shoot fresh weight and shoot length were significantly reduced than the un-inoculated control and the reduction increased with the increase of inoculum’s density. Due to the application of P. lilacinum PLSAU-1, the root length increased than the untreated control and the increased trend was along with increased dose. The highest dose of 1 × 106 CFU/g soil showed the highest root length, where the other two doses showed similar statistical results. Conversely, a significant reduction of root length was found at all inoculated treatments of M. incognita compared to the un-inoculated one. In the case of root fresh weight, the highest dose (1 × 106 CFU/g soil) showed the highest root fresh weight. All the tested inoculum levels showed a significant reduction in root fresh weight than the control. The greater decline of plant growth parameters coupled with an elevated rate of nematode development might be due to the more rapid invasion of roots by larger numbers of J2 and the consequent maturity of adult females. This is possible as the developing giant cell systems and disturbance of the developing xylem lead to change in physiological functions such as nutrient uptake, translocation of nutrients and water from root to plant, and subsequent photosynthesis (Davis et al. 2003).
Obtained results also indicated that the plant growth parameters roughly affected by inoculum density of M. incognita as they decreased with increasing the nematode population. As well, lower plant growth characters were observed when the plants were inoculated by 1600 and 3200 eggs per 100 g soil. Charegani et al. (2012) reported similar results as the inoculation of tomato with inoculum level (40 eggs and J2) of M. incognita or M. javanica per gram of soil reduced shoot length and fresh dry weights markedly, and the reduction was significantly correlated to its inoculum density.
The analytical results showed that all the tested doses of P. lilacinum PLSAU-1 caused significant reductions in gall index rating, number of egg masses per root system, final nematode population, and reproduction factor compared to untreated control (0 CFU/g soil). The highest reduction was observed for the highest (1 × 106 CFU/g soil) dose of bio-agent. In contrast, all the inoculum densities increased the gall index rating and number of egg masses per root system than the un-inoculated control. Inoculating soil with (3200 eggs per 100 cm3 soil) led to a highest gall index development and a number of egg masses per root system. The final nematode population increased with the increase of inoculum level. The highest reproduction factor was observed when the soil was inoculated by 400 eggs/100 cm3 of soil. It showed a statistical similarity with other inoculum levels. The percentage of egg mass colonization and soil colonization was significantly influenced by all tested doses of bio-agents as well as inoculum levels of nematode. The highest percentage of egg mass colonization and soil colonization was recorded when tested with the highest dose (1 × 106 CFU/g soil) of P. lilacinaus. Conversely, the lowest percentage of egg mass colonization and soil colonization were observed at the highest inoculum level (3200 eggs/100 cm3 of soil) of M. incognita (Table 1). The results of the present experiment indicated that the application of P. lilacinum PLSAU-1 suppressed root-knot and increased plant growth parameters.
Two-factor analysis for the plant growth characters (shoot fresh weight, length; root fresh weight, length) showed significant effects on the inoculum level and rates, applied on the brinjal plant. Among the four doses of P. lilacinum PLSAU-1, the rate of (1 × 106 CFU/g soil) enhanced plant growth characters over 400 to 3200 eggs per 100 cm3 soil inoculum level of M. incognita than the untreated inoculated control (Table 2).
The major findings of this study revealed that P. lilacinum controlled the root-knot nematode and M. incognita efficiently increased the plant growth characters. To find out the appropriate dose of P. lilacinum PLSAU-1 against various levels of nematode damage, four fungal doses including 0, 1 × 105, 5 × 105, and 1 × 106 CFU/g soil were applied. Among the different fungal doses, the identified optimum fungal dose was 1 × 106 CFU/g soil, where the highest nematode suppression and improved plant growth was recorded (Table 2). Its existence in the rhizosphere of roots at the penetration time might decrease the number of juveniles that could entrance the roots. This finding is in agreement with Holland et al. (1999) who quantified that P. lilacinum colonized the root and protected its surface from root-knot nematode attacks. It correspondingly reduced the number of viable eggs and juveniles of the second generation during the experimental period. These results confirm the findings of Kiewnick and Sikora (2006). They reported that a single pre-plant application at a concentration of (1 × 106 CFU/g soil) was needed for sufficient biocontrol of root-knot nematode of M. incognita in tomato. In the present study, a significant increase in plant shoots and root length, fresh weight of shoots, and roots was observed by using all doses of P. lilacinum PLSAU-1 compared to untreated control and nematode-inoculated control. Improved character of plant growth by application of P. lilacinum for controlling root-knot nematodes was also reported earlier (Al-Raddad 1995). They tested the effects of Glomus mosseae and P. lilacinum on M. javanica of tomato in a greenhouse experiment. The highest root and shoot length, and fresh and dry weight of root and shoot were achieved when the plants were inoculated by P. lilacinum to control root-knot nematode. Khan et al. (2012) reported that an enhancement in growth and yield of brinjal with biocontrol agents Pochonia chlamydosporia, P. lilacinum, and Trichoderma harzianum owing to suppress gall formation and egg masses. The result of the present study revealed that the brinjal plants inoculated with 3200 eggs of M. incognita showed a significant reduction in shoot and root length, and fresh weight of shoot and root. Reduced plant growth characters by the inoculation of M. incognita were also reported previously by (Ganaie and Khan 2010). The bio-agent significantly reduced the multiplication rate of M. incognita than the control. It proved the potentiality of P. lilacinum PLSAU-1 as a biocontrol agent against M. incognita.
All four doses of P. lilacinum PLSAU-1 were tested, and the lowest gall index development, number of egg masses per root, total nematode population, and reproduction factor were found at 400 and 800 eggs per (100 cm3 soil). For all combination effect of doses and inoculum level, the highest gall index, number of egg masses per root, total nematode population, and reproduction factor were resulted in combination of (1 × 105 CFU/g soil) dose of P. lilacinum PLSAU-1 and 3200 eggs per (100 cm3 of soil) inoculum level of M. incognita. At all the inoculum levels, the gall index, number of egg masses per root, total nematode population, and reproduction factor were significantly reduced by the application of (1 × 106 CFU/g soil) of P. lilacinum PLSAU-1 than the control. However, the maximum reduction of gall index (72%) and number of egg masses per root (84%) was recorded at the dose of 1 × 106 CFU/g soil of P. lilacinum PLSAU-1 against 800 and 400 eggs per 100 cm3 of soil inoculum level, respectively. In this experiment, it revealed a significant interaction between the dose of P. lilacinum PLSAU-1 applied to soil and the inoculum level for the parameters of gall index and number of egg masses per root. A similar effect was demonstrated by Kiewnick et al. (2011), and they reported a strong reduction in the galling index and the number of egg masses per root system after PL251 soil treatment at 2 × 105 CFU/g soil at inoculum density up to 800 eggs/100 cm3 of soil. This result also was confirmed by Aminuzzaman et al. (2018) who mentioned that the root galling index and the final nematode population decreased up to 55.6 and 66.9% for brinjal. In addition, the significant reduction of nematode population (58.5%) was also observed by the same dose against 800 eggs per (100 cm3 of soil) (Table 2). In case of reproduction factor, 81% reduction was found at the 1 × 106 CFU/g soil dose of P. lilacinum PLSAU-1, when pot soil was inoculated by 800 inoculum level per 100 cm3, which was statistically similar to 400 and 1600 inoculum levels, respectively (Table 2). This study indicated that the reduction of M. incognita per plant was depended on the doses of P. lilacinum PLSAU-1. This result is consistent with the results of Kalele et al. (2010) who reported that the pre-planting soil treatment, with the highest dose (0.4 g/10 L soil) reduced 69 and 73% of nematode population in the roots and soil, respectively, than the inoculated control in a tomato plant.
The colonization increased with the increase of a dose of P. lilacinum PLSAU-1 at all the inoculum levels. The highest percentage of egg mass colonization was recorded in 1 × 106 CFU/g soil of P. lilacinum PLSAU-1 against 1600 eggs per 100 cm3 inoculum level, and the highest soil colonization was found at the same doses of bio-agent over 800 eggs per 100 cm3 inoculum level of M. incognita (Fig. 1a, b).
The results of the present study demonstrated that the percentage of egg masses colonized by P. lilacinum PLSAU-1 directly influenced by the fungal density in the soil. This result consistent with the result of Carneiro and Cayrol (1991) who reported that the number of colonized egg masses and the number of non-viable eggs increased with the increase of P. lilacinum density, and the fungus was most effective at a density of 106 spores/g of soil on banana. The P. lilacinum PLSAU-1 was isolated from soil after 2 months of application, which indicated that the fungus survived throughout the growing season and it was compatible with the environmental conditions. Although the empirical result showed that the CFUs were lower than those added at the beginning of the experiment. The decline in CFU numbers, 2–3 months, after the initial application was also reported by several researchers (Kiewnick and Sikora 2003). They revealed that the persistence of entomopathogenic fungi in soil and the relocation of conidia from the superficial to deeper layers are dependent on the presence of antagonistic organisms on the soil properties This result also validated by Rumbos and Kiewnick (2006). They found the decline in fungal densities (CFU numbers) compared to initial densities ranged from 72.3 to 83.8% in the first and in the second experiment in a tomato plant, where they inoculated 2 × 106 CFU/g of dry soil.
The study demonstrated the effects of a dose of P. lilacinum PLSAU-1 to suppress root-knot (M. incognita) damage in infected brinjal roots. The key findings of the present were the strong relationship between doses of bio-agent and inoculum level of the pest. This study suggests that high (1 × 106 CFU/g soil) dose of P. lilacinum PLSAU-1 is necessary for successful control of root-knot nematode M. incognita for all tested inoculum level. However, further research is needed to estimate the interactions between inoculum level of M. incognita, host plant response, and fungus dose to build up an effective dose-response replica for biocontrol fungus.
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We thank to the anonymous reviewers for their kind review and valuable suggestions to improve the earlier version of this manuscript. This research work was supported by the International Foundation for Science, Stockholm, Sweden, [Research Grant Agreement No C/4917-1] and TWAS (The World Academy of Sciences for the advancement of Science in developing countries) [Research Grant No.: 13-246 RG/BIO/AS_/UNESCO FR: 3240277693].
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Authors and Affiliations
College of Plant Science and Technology, Huazhong Agricultural University, Shizishan Street-1, Wuhan, 430070, Hubei, China
Most. Sinthia Sarven
Department of Plant Pathology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka, 1207, Bangladesh
F. M. Aminuzzaman
State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, Wuhan University, 129 Luoyu Road, Wuhan, 430079, Hubei, China
The first author (MSS) of this manuscript performed the experiments of this study and wrote the introduction, methodology, and results parts. The second author (FMA) wrote the discussion and conclusion parts. The third author (MEH) completed the analysis part. All authors read and approved the final manuscript.
This article does not contain any studies with human participants or animals.
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