Introduction

 

Rice blast is a disease caused by Magnaporthe oryzae (Hebert) Barr. This disease is the first recorded of rice (Oryza sativa L.) and it was noted as rice fever disease in China as early as in 1637 (Wang and Valent 2009). Rice blast has spreaded through out in Asia, Latin America and Africa, and is now reported in over 85 countries worldwide (TeBeest et al. 2007). M. oryzae can infect all parts of the plant, which resulted in yield losses in many developing countries in recent years (Wang and Valent 2009). Rice blast has become the most common rice disease due to its wide distribution and high infection level under favourable conditions. Valent (2004) stated that the disease has already caused epidemics in all continents where rice is grown, and yield loss due to blast can be as high as 50% (Gnanamanickam 2009). The effective disease control strategies are needed to reduce or eliminate the use of chemical fungicides, which damaging to the surroundingenvironments and residue in agricultural products. Nanotechnology in agriculture has emerged as a new tool to create and re-structure the materials at the molecular level. Molecular nanotechnology involves in constructing organic materials into defined structures, atom by atom or molecule by molecule (Soutter 2013). The application of nanotechnology in agriculture has gained an interestingattention in recent years (Li et al. 2011). Researchers have actively investigated the synthesis of organic nanomaterials in various types andtested their biological properties (Elibol et al. 2003; Salata 2004). Nanotechnology in agriculture is being explored for crop production (Soutter 2013) and may bepotentially provided the solutions for various challenges faced in agriculture (Ditta 2012). Nanoparticles contain bioactive substances from natural products that can rapidly and effectively penetrate through plant cuticles and tissues and can increase the stability of active compounds to decrease leaching (Perlatti et al. 2013). Thus, it can provide an efficient pest management strategy in agriculture (Rai and Ingle 2012). These can be formulated in colloidal suspension or powder for application (Ditta 2012).

In recent year, the natural products from the fungus Chaetomium spp. reported to be antifungal activity against several plant pathogens (Soytong et al. 2001). Nanoparticles were constructed from crude hexane, ethyl acetate and methanol extracts of Chaetomium globosum KMITL-N0805, whichcoded as Nano-CGH, nano-CGE and nano-CGM, actively inhibited Curvularia lunata (Wakker) Boedijn in rice var. Sen Pidoa, with ED50 values of 1.21, 1.19 and 1.93 ppm, respectively (Tann and Soytong 2016). The findings are demonstrated in vivo tests that nano-CGH, nano-CGE and nano-CGM fromC. Globosum can be controlled leaf spot of rice at 60 days; nano-CGH and nano-CGM decreased disease of 61.54% and nano-CGE decreased disease incidence by 53.83% (Tann and Soytong 2016). Furthermore, Tann and Soytong (2017) reported that nano-product derived from C. cupreum L.M Ames reduced the rice leaf spotranging from 41.7–58.3% compared to the non-treated controlin pot experiment.

The objective of the current research was to evaluate the nanoparticles constructed from natural products of C. cochliodes CTh05 for their biological activities against M. oryzae, which causes rice blast and their application for disease control.

 

Materials and Methods

 

Isolation of pathogens and pathogenicity test

 

The blast specimens were collected from the symptomatic leaves of rice var. RD 57 in rice fields at the Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand on 12 June 2017. Isolation was performed using a tissue transplanting technique (Abed-Ashtiani et al. 2016) and a pure culture was maintained in rice flour agar (RFA) (rice flour 25 g, yeast 2 g and agar 15 g) media. Morphological identification using a binocular compound microscope was done according to the work of Ou (1985). The pathogen was deposited as culture collection No. KMILT 001/2017 at the Biocontrol Research Unit, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand.

 

Antagonistic fungus

 

C. cochliodes CTh05, used in the current study, has been reported by Phonkerd et al. (2008) to produce four dimeric spiro-azaphilones; (cochliodones A to D), two azaphilones; (chaetoviridines E and F) and an epi-chaetoviridin A, which expressed antimicrobial activity against malaria disease (Plasmopara falciparum Welch), tuberculosis (Mycobacterium tuberculosis Zopf) and cancer cell lines. C. cochliodes CTh05 was cultured in potato dextrose agar (PDA) and incubated at room temperature (27–30°C). The morphological identification was performed according to Arx et al. (1986) and Soytong (1989).

 

Molecular identification

 

The fungal genomic DNA was separately extracted from M. oryzae PO1 and C. cochliodes CTh05. Each fungus was cultured for 3 d in potato dextrose broth (PDB). The genomic DNA was done from freeze-dried mycelia using the modified cetyl trimethyl ammonium bromide (CTAB) method. The mycelia were cleaned with 25 mM ethylene diamine tetraacetic acid (EDTA) by centrifugation at 14 000 rpm at 4°C for 5 min, then, 100 mg of mycelia were crushed in liquid nitrogen, and lysed in CTAB buffer containing β-mercaptoethanol (2 µL). The lysate was extracted with an equal volume of chloroform/isoamyl alcohol (24:1) and then centrifuged at 14 000 rpm at 4°C, for 5 min then, 2 µL of Rnase (20 µg mL) was added to the aqueous phase and incubated at 37°C for 30 min. The samples were mixed with 50 µL of 10% CTAB and centrifuged. The pellets were washed twice with 70% and 95% ethanol and dissolved in 100 µL TE (Tris-EDTA) buffer at 37°C overnight. The quality and quantity of extracted DNA were monitored by electrophoresis on a 1% agarose gel. Quantification of the DNA was performed by comparing the intensity of the bands to known dilutions of lambda phage DNA. Polymerase chain reaction (PCR) was done to amplify the internal transcribed spacer (ITS) ribosomal DNA regions using the universal primers ITS1 and ITS4, according to the method of White et al. (1990).

The amplified products were sequenced and aligned with sequencing in the GeneBank by the basic local alignment search tool (BLAST) (Altschul et al. 1997) at the National Centre for Biotechnology Information (NCBI) database. The sequences of closely related organisms were downloaded to construct the phylogenetic trees, which were aligned through CLUSTALW using MEGA version 6.0 software (Tamura et al. 2007). The phylogenetic tree was done according to the neighbour-joining method.

 

Biculture test

 

The biculture test was conducted by following the method described by Soytong and Quimio (1989). The experimental design was done by completely randomized design (CRD) with four repeated experiments. C. cochliodes CTh05 and M. oryzae were transferred separately on agar plugs (0.3 cm diameter) to RFA at opposite sites in bi-culture plates. C. cochliodes CTh05 and the rice blast pathogen were separately cultured and each isolate on RFA served as the control. All plates were maintained at room temperature for 30 d. Data collection were recorded on colony diameter (cm), number of spores and were computed analysis of variance (ANOVA) by the Statistical Package for Social Sciences (IBM SPSS Statistics, ver. 21.0) software (Titone et al. 2015). Significance was declared at P ≤ 0.05 and 0.01.

 

Bioactivity tests of crude metabolites from C. cochliodes CTh05

 

Crude extracts from C. cochliodes CTh05 were cultured in PDB at room temperature (30°C) for 30 d. The dried fungal biomass culture was separatelyextracted with hexane, ethyl acetate and methanol following the method described by Phonkerd et al. (2008). The experiment was designed as a two-factor factorial experiment with a CRD and four replications. Factor A represented the crude extracts hexane, ethyl acetate and methanol, and factor B represented theconcentrations including 0, 10, 50, 100, 500 and 1000 ppm. A culture agar plug of 3 mm was transferred to the middle of RFA plate in each treatment and then incubated at room temperature (27–30°C) for 15 d. The data were presented as the colony diameter (cm), and the number of spores was determined using a hemocytometer. The data were analysed using ANOVA by SPSS software ver. 21.0 (Titone et al. 2015). Significance was declared at P ≤ 0.05 and 0.01. The effective dose (ED50) was done using the probit analysis program (Titone et al. 2015).

 

Bioactivity tests of crude metabolite nanoparticles from C. cochliodes CTh05

 

The morphological characteristics of the nano-CCoH, nano-CCOE, and nano-CCoM were viewed under a scanning electron microscope. The crude extracts from C. cochliodes CTh05, including crude hexane, ethyl acetate and methanol extracts, were separately incorporated into polylactic acid-based nanoparticles through electrospinning, following the method described by Dar and Soytong (2014) and Tann and Soytong (2016) to yield nanoparticles from crude hexane, ethyl acetate and methanol extrtacts of C. cochliodes CTh05, coded as nano-CCoH, nano-CCoE and nano-CCoM. The nanoparticle products were collected and stored in capped bottles after electrospinning. The characteristics of the nano-CCoH, nano-CCoE and nano-CCoM were observed by the naked eye and viewed under a scanning electron microscope and the properties were analysed by Fourier-transform infrared spectroscopy (FTIS).

The nano-CCoH, -CCoE and -CCoM were tested for their abilities to inhibit the rice blast pathogen. The experiment was performed using a two-factor factorial CRD with four replications. Factor A represented the type of nanoparticles and factor B the concentrations (0, 3, 5, 7, 10 and 15 ppm). The experiment was repeated four times. The data presented as colony diameter (cm) and the number of spores. Statistical significance was determined using ANOVA by SPSS software, ver. 21.0 (Titone et al. 2015). Significance was declared at P ≤ 0.05 and 0.01. The effective dose (ED50) was calculated using the probit analysis program (Titone et al. 2015).

In vivo nanoparticles constructed from C. cochliodes CTh05 against rice blast disease

 

The experimental design was used as randomized complete block (RCB) with four replicates. The treatments were performed as follows: Treatment 1 was the non-inoculated control, Treatment 2 was the inoculated control, Treatment 3 was the nanoparticles from a crude extractmixture of C. cochliodes CTh05 at 10 ppm, and Treatment 4 was the chemical fungicide (tricyclazole) at the recommended rate of 2.25 g L-1. The data were recorded as the fresh and dry weight of the stems at 90 d and computed analysis of variance. Mean comparison in each treatment was done by SPSS software, ver. 21.0, and significance was declared at P ≤ 0.05 and 0.01. Plants were assigned a disease index at 7 d post-inoculation using a scale of 0–9 (modified from Xia et al. 1993) where 0 = no infection, 1 = small brown spot infection < 1 mm; 2 = small rounded spot infection < 2 mm; 3 = small spot infection with open centres < 3 mm; 4 = lesions with expanded open centres > 3 mm on < 10% of the leaf area; 5 = lesions with expanded open centres on 10–25% of the leaf area; 6 = lesions with expanded open centres on 26–50% of the leaf area; 7 = expanded lesions with open centres on 51–75% of the leaf area; 8 = expanded lesions with open centres on 76–90% of the leaf area; 9 = expanded lesions with open centres on > 90% of the leaf area.

 

In vivo nano-particles from C. cochliodes CTh05 against rice blast disease

 

This experiment was designed as a RCBD with four replications. Nanoparticles derived from hexane, ethyl acetate and methanol crude extracts from C. cochliodes CTh05 were separately appliedat a concentration of 7 ppm to the rice seedlings inoculated with M. oryzae PO1. The treatments were done as follows: non-inoculated control (T1), inoculated with M. oryzae PO1 (T2), nano-CCoH (T3), nano-CCoE (T4), nano-CCoM (T5) and tricyclazole (T6). The disease index (DI) was evaluated as described above and disease reduction (DR) was calculated as follows:

 

 

Results

 

Isolates of rice blast pathogen and Chaetomium spp.

 

The pathogen isolated from symptomatic leaves of rice var. RD57 was morphologically identified as M. oryzae isolate PO1 (Fig. 1). The fungus was cultured on RFA which covered the plate (9 cm) in 10 d. The mycelia were observed to be septate and hyaline, producing conidiophores and three-celled conidia. C. cochliodes CTh05, from a previous study by Phonkerd et al. (2008), was cultured on PDA for 3 weeks and was olive-green to brown, producing perithecia and subglobose asci; one ascus containing eight ascospores (Fig. 1).

 

Fig. 1: Magnaporthe oryzae PO1, A = pure culture in RFA; B = conidium; C = mycelia and conidia and Chaetomium cochliodes CTh05, D = Colony, E = Perithecia, F = Asci and ascospores

 

Description: Description: Description: Description: C:\Users\apple\Desktop\PO1.png

 

Fig. 2: Phylogenetic tree of Magnporthe oryzae from GenBank, including Magnporthe oryzae PO1, constructed based upon the distance-based analysis of the ITS1 and 5.8S regions of rDNA. The numbers at the branches indicate the percentage of bootstrap values after 1000 replications. The outgroup taxon is Sordaria fimicola

 

Description: Description: Description: Description: C:\Users\apple\Desktop\CTh05.png

 

Fig. 3: Phylogenetic tree of C. cochliodes from GenBank, including C. cochliodes CTh05, constructed based upon the distance-based analysis of the ITS1 and 5.8S regions of rDNA. The numbers at the branches indicate the percentage of bootstrap values after 1000 replications. The outgroup taxon is Colletotrichum gloeosporioides

Molecular phylogenic identification was performed to confirm the species. The phylogenetic treewasclearly identified the rice blast pathogen as M. oryzae MH590369, based upon the GeneBank database (Fig. 2). Data from the GeneBank reliably confirmed CTh105 as C. cochliodes MH590621 (Fig. 3). The pathogenicity of M. oryzae isolate PO1 to rice var. RD57 proved to be the blast pathogen. The inoculated wounds exhibited lesions inroundish to elongated grey necrotic spots, approximately 2–5 mm in diameter within 10 d.

Biculture test

 

M. oryzae isolate PO1 was inhibited by C. cochliodes CTh05. The colony diameter of C. cochliodes CTh05 averaged 4.4 cm in bicultureplate whereas the control platewas 9.0 cm. The colony growth inhibition was 52% after 10 d but when the incubation period was extended to 30 d, the colony grew over the pathogen and inhibition averaged above 90%.

 

Characterization of the nano-particles

 

The nanoparticles nano-CCoH, nano-CCoE and nano-CCoM, loaded with crude extracts from C. cochliodes CTh05, were visually characterized. Nano-CCoH, nano-CCoE and nano-CCoM were white, yellow and light yellow in colour, respectively (Fig. 4). Interestingly, the scanning electron images showed the range of particles size of nano-CCoH, nano-CCoE and nano-CCoM ranged between 567–611, 422–566 and 415–472 nm, respectively (Fig. 4).

 

Bioactivity test of crude metabolites from C. cochliodes CTh05

 

The results demonstrated that CCoE resulted in the highest spore inhibition of 88%, followed by CCoM and CCoH, which resulted in a spore inhibition of 81 and 68%, respectively, in 12 d at 1000 ppm (Table 1). The fungal metabolites of CCoH, CCoE and CCoM exhibited active antifungal activity against the M. oryzae isolate PO1 with ED50 values of 85, 144 and 374 ppm (Table 1).

 

Bioactivity tests of crude metabolite nanoparticles from C. cochliodes CTh05

 

The nano-CCoH, nano-CCoE and nano-CCoM at 15 ppm showed significantly inhibited spore production (P ≤ 0.01) in comparison to the non-treated control (0 ppm). Nano-CCoE resulted in significantly (P ≤ 0.05) greater spore inhibition in comparison to the non-treated control. Nano-CCoE inhibited spore production significantly (P ≤ 0.05; 68%), followed by nano-CCoM (47%) and nano-CCoH (34%) in 12 d (Table 2). Nano-CCoE, nano-CCoM and nano-CCoH inhibited M. oryzae PO1 (rice blast) with ED50 values of 9.47, 16.51 and 33.41 ppm, respectively (Table 2). It was observed that the spores were abnormally shaped, and cells were broken after treatment with nanoparticles of C. cochliodes CTh05; in contrast, the spores were normally shaped in the non-treated control (Fig. 5).

 

In vivo nanoparticles constructed from C. cochliodes CTh05 against rice blast disease

 

The result revealed that the blast incidence caused by the M. oryzae isolate PO1 was significantly reduced (P ≤ 0.01) by 38%, after the application of nanoparticles from C. cochliodes CTh05, followed by the chemical fungicide treatment (tricyclazole), which reduced blast incidence by 29% when compared to the inoculated controlwith M. oryzae PO1 (Table 3). In the in vivo experiment, all treatments resulted in significant differences (P ≤ 0.01) in plant height at 15 d post-treatment (Fig. 6). The application of nanoparticles of C. cochliodes CTh05 resulted in the greatest plant height (84.7 cm), which was significant at P ≤ 0.01, followed by the chemical fungicide (tricyclazole), which resulted in a plant height of 74.9 cm; the non-inoculated control and the control inoculated with M. oryzae exhibited heights of 77.7 and 77.6 cm, respectively. The application of nanoparticles from C. cochliodes CTh05 and tricyclazole were not significant differences in stem fresh weight, 64.7 and 66.7 g, respectively, but they were significantly differed (P ≤ 0.01) from the non-inoculated control and the inoculated with M. oryzae, 57.2 and 45.5 g, respectively. The root fresh weight showed the highest (79.4 g) in plants which treated with nanoparticles of C. cochliodes CTh05, followed by the non-inoculated control, the inoculated with M. oryzae, and the plants treated with tricyclazole treatments: 66.6, 58.0 and 44.2 g, respectively (Table 3). The nanoparticles of C. cochliodes resulted in significantly (P ≤ 0.01) higher stem dry weight (14.1 g) compared with tricyclazole (11.1 g) and inoculation with M. oryzae alone 12.0 g (Table 3). The root dry weight showed the highest after treatment with tricyclazole (11.3 g), followed by the nanoparticles of C. cochliodes (10.5 g) and inoculated with M. oryzae isolate PO1 alone (6.4 g).

 

In vivo nano-particles from C. cochliodes CTh05 against rice blast disease

 

The results demonstrated that 15 d after the treatments, nano-CCoM resulted in (P ≤ 0.01) greater plant height (43.0 cm) compared with nano-CCoH and nano-CCoE, which resulted in a plant height of 41.0 and 40.5 cm, respectively, and followed by tricyclazole 38.5 cm, meanwhile, the inoculated control showeda plant height of 38.6 cm (Table 4 and Fig. 7). However, 30 d after treatment, plant height did not significantly differ among plants treated with nano-CCoM, nano-CCoE and tricyclazole; for these groups, plant heights were 77.8, 76.3 and 75.0 cm, respectively and followed by the nano-CCoH (62.3 cm) treatment. The inoculated control had a plant height of 53.8 cm. The blast incidence was reduced after treatment with nano-CCoM (60%) more than nano-CCoE (58%), tricyclazole (56%) or nano-CCoH (50%).

Table 1: The effects of fungal metabolites of C. cochliodes CTh05 against M. oryzae PO1 at 12 d

 

Metabolites

Concentration (ppm)

Colony diameter (cm)

Growth inhibition (%)

Number of spores  (105)

Spore Inhibition (%)

ED50(ppm)

CCoH

0

5.00a

-

20.75a

-

 

10

4.88bc

2.25ij

20.25a

2.56j

 

50

4.73d

5.25gh

17.25b

16.86h

374.43

100

4.43f

11.25e

14.25c

31.23g

 

500

3.91i

21.75b

10.00e

52.18de

 

1000

3.68j

26.25 a

6.75fg

67.57c

 

CCoE

0

5.00a

-

20.75a

-

 

10

4.94ab

1.00j

17.25b

16.70h

 

50

4.82c

3.50i

12.75cd

38.64f

85.87

100

4.67d

6.50g

9.00ef

56.69d

 

500

4.02h

19.50c

5.00gh

76.01b

 

1000

3.64j

27.00a

2.50h

88.03a

 

CCoM

0

5.00a

-

20.75a

-

 

10

4.86c

2.75i

18.75ab

9.68i

 

50

4.73d

4.00hi

14.00c

32.58g

144.23

100

4.53e

9.25f

11.00de

46.95e

 

500

4.11g

17.75d

7.00fg

66.40c

 

1000

3.91i

21.75b

4.00h

80.74b

 

C.V.(%)

0.74

7.22

10.79

6.27

-

Means followed by a common letter are not significantly different by DMRT at P ≤ 0.05

 

Table 2: The effects of nano particles derived from C. cochliodes CTh05 against M. oryzae PO1 at 12 d

 

Nano-particles

Concentration (ppm)

Colony diameter (cm)

Growth inhibition (%)

Number of spores (105)

Spore Inhibition (%)

ED50 (ppm)

Nano-CCoH

0

5.00a

-

69.00a

-

 

3

4.96ab

0.75e

67.00ab

2.78i

 

5

4.88bc

2.25de

56.00bcd

18.98f

33.41

10

4.82cd

3.50cd

50.75cd

26.63e

 

15

4.76d

4.75c

45.00de

34.87cd

 

Nano-CCoE

0

5.00a

-

69.00a

-

 

3

4.93ab

1.25e

60.00abc

13.23fg

 

5

4.87bc

2.50de

49.00cd

28.99de

9.47

10

4.78d

4.25cd

36.00e

47.99b

 

15

4.58e

8.25b

22.00f

68.26a

 

Nano-CCoM

0

5.00a

-

69.00a

-

 

3

4.87bc

2.50de

64.00ab

7.36gh

 

5

4.77d

3.75cd

51.25cd

26.00e

16.51

10

4.56e

8.75b

44.00de

36.40c

 

15

3.88f

22.25a

36.00e

47.97b

 

C.V.(%)

1.18

25.04

11.03

13.91

-

Means followed by a common letter are not significantly different by DMRT at P ≤ 0.05

Table 3: Effect of crude extract mixture nano-particles on plant height, fresh and dry weight of stems and roots and disease reduction of blast in rice var. RD 57

 

Treatments

Plant height

(15 d) (cm)

Plant height

(30d) (cm)

Stem

Fresh weight (g)

Root

Fresh weight (g)

Stem

Dry Weight (g)

Root

Dry Weight

(g)

Disease

Reduction (%)

T1(Non-inoculated control)

62.0a

77.74b

57.23b

66.55b

17.81a

8.82b

-

T2 (Inoculated with M. oryzae) 

62.6a

77.58b

45.53c

58.03c

12.01c

6.42b

-

T3(M. oryzae + nanoparticles of C. cochliodes )

63.3a

84.67a

64.70a

79.41a

14.05b

10.51a

37.5

T4 (M. oryzae + Tricyclazole )

62.1a

74.91b

66.66a

44.20d

11.12cd

11.28a

29.1

CV(%)

2.52

12.48

22.82

29.87

29.29

28.96

-

Means followed by a common letter are not significantly different by DMRT at P ≤ 0.05

 

Table 4: Effect of nanoparticles derived from hexane, ethyl acetate and methanol extraction plant height and disease reduction (%) on rice var. RD 57

 

Treatments

15 (d)

30 (d)

Disease reduction (%)

T1 (Non-inoculated control)

38.70c

54.50c

-

T2 (Inoculated with M. oryzae)

38.60c

53.75c

-

T3 (Nano-CCoH)

41.00ab

62.25b

50.2

T4 (Nano-CCoE)

40.50b

76.25a

57.5

T5 (Nano-CCoM)

43.00a

77.75a

59.8

T6 (Tricyclazole)

38.50c

75.00a

55.5

Means followed by a common letter are not significantly different by DMRT at P ≤ 0.05

 

Discussion

 

In this study, C. cochliodes CTh05 demonstrated activity against the M. oryzae isolate PO1, which causes rice blast. The fungal isolate that caused blast symptoms on rice var. RD57 was morphologically and molecularly identified as M. oryzae. Morphology and molecular techniques confirmed the identity of C. cochliodes CTh05. M. oryzae isolate PO1 found to be a virulent isolate causing blast of rice var. RD57. Biculture tests showed that C. cochliodes CTh05 inhibited the growth of M. oryzae PO1. A previous report by Soytong (2014) stated that C. cochliodes proved to be antagonistic to Drechslera oryzae (brown leaf spot of rice var. Pittsanulok 2). C. cochliodes CTh05 significantly inhibited colony growth and spore production of the tested pathogen in biculture tests, as reported by Tann and Soytong (2017) for C. cupreum CC3003 inhibiting C. lunata (leaf spot of rice).

 

Fig. 4: Nanoparticles from C. cochliodes CTh05 (upper part: A= nano-CCoH, B = nano-CCoE, C = nano-CCoM) and scanning electron microscopy of nano-particles (lower part: D = nano-CCoH, E = nano-CCoE and F =nano-CCoM)

 

 

Fig. 5: Normal spores of the rice blast pathogen (A) and abnormal spores (B) of the M. oryzae isolate PO1 after treatment with nanoparticles derived C. cochliodes CTh05

 

Fig. 6: Testing the capacity for nanoparticles derived from C. cochliodes CTh05 to inhibit rice blast, T1 = non-inoculated control, T2 = inoculated with M. oryzae, T3 = M. oryzae + nanoparticles of C. cochliodes, and T4 = M. oryzae + Tricyclazole

 

 

Fig. 7: Testing the capacity of nanoparticles derived from C. cochliodes CTh05 in inhibiting rice blast 15 days after treatments, non-inoculated control (T1), inoculated with M. oryzae (T2), nano-CCoH (T3), nano-CCoE (T4), nano-CCoM (T5) and tricyclazole (T6)

The fungal metabolites of C. cochliodes CTh05 (CCoH, CCoE and CCoM) expressed antifungal activity against M. oryzae isolate PO1 and inhibited spore production with ED50 values of 374, 85, 144 ppm. Soytong (2014) reported that metabolites from C. cochliodes suppressed the spore production of D. oryzae (brown leaf spot of rice) significantly, with an ED50 value of 66.45 ppm. Tann and Soytong (2017) reported that the hexane-crude extract, ethyl acetate-crude extract and methanol-crude extract of C. cupreum C3003 inhibited the spore production of C. lunata (leaf spot of rice) with ED50 values of 6.41, 0.83 and 7.81 ppm, respectively. C. cochliodes CTh05, which was tested in the current study, was previously reported to produce chaetoviridines E, chaetochalasin A and 24(R)-5a, 8a-epidioxyergosta-6-22-diene-3b-ol which those compounds inhibited Plasmodium falciparum. The cochliodones C, chaetoviridines E, chaetoviridines F and chaetochalasin A exert antimycobacterial activity. Moreover, chaetoviridines E and F inhibitedcytotoxicity against the KB, BC1 and NCI-H187 cell lines (Phonkerd et al. 2008). The current study revealed that the fungal metabolites from C. cochliodes CTh05 suppressed M. oryzae PO1, which causes rice blast.

Nanoparticles derived from C. cochliodes CTh05 (nano-CCoH, nano-CCoE and nano-CCoM) demonstrated to inhibit M. oryzae PO1 with ED50 values of 9, 16 and 33 ppm, respectively. C. cochliodes CTh05 has been found to produce the active compoundsagainst human pathogens (Phokerd et al. 2008). Those active compounds may possible serve as a control mechanism.

The crude extracts derived from C. cochliodes CTh05, CCoH, CCoE and CCoM found to inhibit M. oryzae at ED50 values of 374, 85 and 144 ppm, respectively. The nanoparticles of nano-CCoH, nano-CCoE and nano-CCoM gave high potential at low application, with ED50 values of 33, 9 and 16 ppm, respectively. It is suggested that applying nanoparticles would be less costly than metabolites. A similar report by Tann and Soytong (2016) stated that nano-CGH, nano-CGE and nano-CGM derived from C. globosum KMITL-N0805 exhibited antifungal activity against C. lunata (leaf spot of rice) with ED50 values of 1.21, 1.19, and 1.93 ppm, respectively.

The current study revealed that the spores of M. oryzae exhibited abnormal morphology after treatment with nanoparticles from C. cochliodes CTh05. It was also reported by Tann and Soytong (2017) that nanoparticles from C. globosum can disrupt and distort the spores of C. lunata and cause a loss of pathogenicity. Singh et al. (2015) reported that nanotechnology in agriculture is being revolutionized by innovative new techniques for disease control. The current research revealed that nanoparticles constructed from fungal metabolites are promising for plant disease control. Dar and Soytong (2014) reported that the fungal metabolites from C. globosum and C. cupreum, generated to be nanomaterials at the averaged size of 241 and 171 nm, respectively. Tann and Soytong (2016) also reported that nanoparticles from C. globosum can disrupt and distort the pathogen cells and cause a loss of pathogenicity. Nano-CCoH, nano-CCoE and nano-CCoM constructed from C. cochliodes CTh05 may help to increase the efficiency of application at lower doses to inhibit rice blast pathogen as stated by Sharon et al. (2010).

In the current work, blast incidence in rice seedlings treated with nano-particles constructed from a crude extract mixture at 10 ppm was reduced by 38%, and followed by the chemical fungicide treatment (tricyclazole) which reduced blast incidence by 29%. Similar results were reported by Soytong (2014), who stated that bioproducts from C. cochliodes CTh05 can control the rice leaf spot caused by C. lunata, but at a high application rate of 50 g 20 L-1 of water.

 

Conclusion

 

In vivo evaluation of nano-CCoM, nano-CCoE and nano-CCoH constructed from crude hexane, ethyl acetate and methanol extracts of C. cochliodes CTh05 reduced blast incidence at concentrations of 7 ppm. It suggested that application of nanoparticles constructed from each crude extract (nano-CCoM, nano-CcoE and nano-CCoH) reduced blast incidence at a lower rate of application than nanoparticles derived from a crude extract mixture from C. cochliodes CTh05. Further research should investigate these nanoparticles as elicitors for rice immunity to blast disease.

 

Acknowledgments

 

I would like to give special thanks to Biocontrol Research Laboratory, Faculty of Agricultural Technology for offering all facilities during my research investigation. I would be acknowledged to King Mongkut’s Institute of Technology Ladkrabang (KMITL) to offer of a Ph.D. Scholarship, and Thailand Research Fund to support a part of my research project. Special thanks conveyed to Department of Chemistry, Faculty of Science, Khon Khan University, Thailand for chemistry work.

 

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