In Vitro Efficacy of Botanicals against Rice Blast Pathogen Magnaporthe oryzae oryzae

Rice is the most staple cereal crop of Bangladesh and rice blast caused by Magnaporthe oryzae oryzae (MoO) has become a major factor limiting rice yield in Bangladesh and throughout the world. Eight botanicals extracted both in water and ethanol namely Kalijira (Nigella sativa), Turmeric (Curcuma longa), Ginger (Zingiber officinalis), Garlic (Allium sativum), Onion (Allium cepa), Neem (Azadirachta indica), Allamanda (Allamanda cathartica) and Aloevera (Aloe vera) were tested against MoO in vitro in the Mycology Laboratory, Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. All the botanicals significantly reduced radial growth of the tested pathogen. Maximum mycelia growth inhibition of MoO was achieved with water extract of turmeric (1:1 w/v) and ethanol extracts of neem (1:4 w/v) with 86.57% and 92.62% mycelia growth inhibition at 14 DAI, respectively.


Introduction
Rice (Oryza sativa L.) is consumed as a staple food for half of the world's population [1]. In Bangladesh, rice is the most staple cereal crop and central to Bangladesh's economy, accounting for nearly 20 percent of gross domestic product (GDP) and providing about one-sixth of the national income of Bangladesh [2].
But it is a great concern that rice blast caused by Magnaporthe oryzae has become a major factor limiting rice yield throughout the world [3] [4] [5].
(2021) In Vitro Efficacy of Botanicals against Rice Blast Pathogen Magnaporthe oryzae oryzae. American Journal of Plant Sciences, tional capacity of rapidly changing its genetic makeup resulting in new pathogenic variants (races) [7] [8]. It is the causal agent of rice blast, one of the most devastating diseases of rice (Oryzae sativa L.) observed in most of the rice growing countries across the world [9]. The pathogen can cause infection on leaves, stems, peduncles, panicles, seeds and even roots. This disease is the potential threat that may cause crop failure and yield loss. Thus it has been ranked among the most important rice diseases. Pyricularia oryzae (Po) was isolated from infected leaf and panicle and identified based on cultural characteristics and conidia morphology and mycelia growth of Pyricularia isolates varied significantly with fair to excellent sporulation ability [10].
To initiate rice blast, the MoO has evolved a unique mechanism for conidium attachment to rice leaf surfaces. The disease can be severe during periods of cool temperatures and high moisture, while conidia do not germinate under direct sunlight [5]. Cloudy overcast weather and dew encourage blast spread. Conidia remain viable during winter even under snow. Infected host residue is the most important source of the primary inoculum causing epidemics initiation [11]. Survival of the fungus was greatly reduced during winter, but during spring, sporulation of the fungus occurred on plant debris [12]. Dissemination of the fungus also involves a wide range of alternative host plants [13]. In temperate regions, infested rice seed, straw, and residues have been implicated as the most important overwintering sources of primary inoculum, although their impacts on initial disease development and distribution is not fully understood [14]- [19] and [5].
However, the frequent use of fungicides on crops may cause hazards to human beings, plant health, beneficial micro-organisms, and develop fungicide resistance into the pathogens and residual toxicity in plant parts. On the other hand, some botanical extracts have proved to be most secure and have no adverse impact on environment [22] [23]. Plant extracts like garlic juice successfully reduced the infection caused by Magnaporthe sp., on rice [24]. Mycelia growth of rice blast fungus was also significantly reduced by water and ethanol leaf extracts, and oil extract of neem seed [25]. The purpose of this study was to evaluate comparative in vitro efficacy of water and ethanol extracts of botanicals against MoO causing rice blast.

Experimental Site
The experiment was conducted in the Mycology Laboratory, Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh.

Experimental Period
The experiment was conducted during the period from June 2018 to December 2019.

Preparation of Botanical Extracts
Botanicals extracted with either water or ethanol in different concentrations were used to understand the efficiency of the botanicals those are: 1:4 (w/v) = 25 g botanical in 100 ml either water or ethanol   1:2 (w/v) = 50 g botanical in 100 ml either water or ethanol. 1:1 (w/v) = 100 g botanical with 100 ml either water or ethanol.

Water Extract of Botaticals
In case of water extract of botanicals, 25 g, 50 g and 100 g botanicals were crushed in 100 ml water separately, grinded with mortar and pestle in water temperature (22˚C) and in vitro condition for 10 mins. Then the extracted juice were seived and taken in conical flask. Then different concentrations were applied in PDA (Potato Dextrose Agar) culture plates and then 15 days old mycelia discs of MoO were cut using disc cutter and placed in the middle of the petridish. The experiments were done in vitro following poisoned food technique [26]. Radial mycelia growth was recorded in 7, 10 and 14 days after inoculation (DAI).

Ethanol Extracts of Botanicals
Incase of botanical ethanol extracts, 25 g, 50 g and 100 g botanicals grinded with mortar and pestle, then mixed with 100 ml ethanol separately. The mixtures were then kept overnight in beakers covered with aluminium foil for increasing the extraction efficiency and next day extracted juice were seived and taken in conical flask. Then different concentrations were applied in PDA (Potato Dextrose Agar) culture plates and then 15 days old mycelia discs of MoO were cut using disc cutter and placed in the middle of the petridish. The experiments were done in vitro following poisoned food technique [26]. Radial mycelia growth was recorded in 7 and 14 DAI.

Experimental Design and Statistical Analysis
The experiment was done following Complete Randomized Design (CRD) with three replications and statistical analysis was done using Statistix10 software. Treatment means were compared by Duncan's Multiple Range Test (DMRT). Least significant differences (LSD) were calculated using significant level at P = 0.05.

Results and Discussions
The present study was conducted to evaluate the efficacy of botanical extracts against a virulent isolate of Magnaporthe oryzae oryzae MoO19 [27]. The isolate was identified based on three celled pyriform conidia ( Figure 2). The results with discussion of the experiments conducted on these lines are presented in this chapter.   Each value is the mean of three replications. In a column, figure having same letter(s) do not differ significantly at 5% level by DMRT.

Efficacy of Botanical Water Extracts in Controlling Radial Mycelia Growth of MoO in Vitro
Turmeric in 1:1 (w/v) that is 8.33 mm at 7 DAI. Maximum growth inhibition of MoO was achieved with turmeric 1:1 (w/v) with 82.15% mycelia growth inhibition which was significantly different and superior to rest of the treatments ( Figure 3). The next botanicals in order of merit were turmeric 1:2 (w/v) with 74.29% growth inhibition compared to control. The next botanicals in order of merit were garlic 1:2 (w/v), ginger 1:1 (w/v), onion 1:2 (w/v) and allamonda 1:1 (w/v) with 65.72%, 62.87%, 61.43% and 61.43% mycelia growth inhibition respectively of the test fungus as compared to control. After that in order of merit were kalijira 1:1 (w/v), ginger 1:2 (w/v), garlic 1:4 (w/v) and onion 1:4 (w/v) with 59.29% growth inhibition of the test fungus as compared to control. After in that order of merit were onion 1:1 (w/v), aloe vera 1:1 (w/v) and turmeric 1:4 (w/v) with 57.14%, 55.71% and 55% growth inhibition of the test fungus as compared to control. Aloe Vera 1:4 (w/v) with 7.86% inhibition was found to be the least effective botanicals. Graphical representation was shown in Figure 4.   (Table 2). Lowest mycelia growth was recorded in Turmeric in 1:1 (w/v) that is 9 mm at 10 DAI. Maximum growth inhibition of MoO was achieved with turmeric 1:1 (w/v) with 83.12% mycelia growth inhibition which was significantly different and superior to rest of the treatments (Figure 3). The next botanicals in order of merit were turmeric 1:2 (w/v), garlic 1:2 (w/v) and ginger 1:1 (w/v) with 70%, 68.12% and 67.50% mycelia growth inhibition respectively of the test fungus as compared to control. After that in order of merit were kalijira 1:1 (w/v), ginger 1:2 (w/v) and allamonda 1:1 (w/v) with 64.37% growth inhibition of the test fungus as compared to control. After in that order of merit was onion 1:2 (w/v) with 62.50% growth inhibition of the test fungus as compared to control. After in that in order of merit were onion 1:1 (w/v), onion 1:4 (w/v) and turmeric 1:4 (w/v) with 59.37%, 58.75% and 58.75% inhibition of growth of the test fungus as compared to control. After in that of merit order ginger 1:4 (w/v) and aloe vera 1:2 (w/v) with 55% growth inhibition of the test fungus as compared to control. Kalijira 1:4 (w/v) with 2.5% inhibition was found to be the least effective botanicals in in vitro. Graphical representation was shown in Figure 4.

3) In Vitro Mycelia Growth of MoO at 14 DAI
In vitro mycelia growth of MoO on different botanical treatment at 14 DAI was found significantly different ( Table 2). Lowest mycelia growth was recorded in Turmeric in 1:1 (w/v) that is 9 mm at 14 DAI. Maximum growth inhibition of MoO was achieved with turmeric 1:1 (w/v) with 86.57% mycelia growth inhibition which was significantly different and superior to rest of the treatments (Figure 3). The next botanicals in order of merit was turmeric 1:2 (w/v) with 73.13% mycelia growth inhibition of the test fungus as compared to control. After that in order of merit were ginger 1:2 (w/v) and ginger 1:1 (w/v) with 71.64% and 71.15% growth inhibition respectively of the test fungus as compared to control. After in that order of merit were turmeric 1:4 (w/v) and allamonda 1:1 (w/v) with 69.15% and 67.16% growth inhibition respectively of the test fungus as compared to control. Kalijira 1:4 (w/v) with 11.45% inhibition was found to be the least effective botanicals in in vitro. Graphical representation was shown in Figure 4.
Curcuma longa L. (Zingiberaceae family) and its polyphenolic compound curcumin have been subjected to a variety of antimicrobial investigations due to extensive traditional uses and low side effects. Antimicrobial activities for curcumin and rhizome extract of C. longa against different bacteria, viruses, fungi, and parasites have been reported [28]. According to [29] fungicidal activity of turmeric rhizome-derived materials was tested using a whole plant method in vivo against Botrytis cinerea, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia oryzae, and Rhizoctonia solani. Our result was accordance with [30] who studied on a satisfactory potential of turmeric as a natural pesticide for possible use in crop protection and thus a highly promising future towards this direction, that is, the possibility of effective control of certain pests of agricultural importance with the use of turmeric products as a cheap and more environment friendly alternative to chemical pesticides already used for the same purpose. Essential oils and oleoresin from Piper nigrum, Coriander sativum and Curcuma domestica were isolated and tested for their effect on Magnaporthe oryzae in rice [31].

Mycelia Growth of MoOIn Vitro
Among those eight botanicals in 1:4 (w/v) at 7 DAI, highest mycelia growth was recorded in Aloe Vera (43 mm) of the test fungus compared to control (46.67 mm) and lowest mycelia growth (19 mm) in garlic and onion extracts was recorded (Table 3).
Among those eight botanicals in 1:4 (w/v) at 10 DAI, highest mycelia growth was recorded in kalijira (52 mm) of the test fungus compared to control (53.33 mm) and lowest mycelia growth (22 mm) in turmeric and onion was recorded (Table 3).
Among those eight botanicals in 1:4 (w/v) at 14 DAI, highest mycelia growth was recorded in kalijira (59.33 mm) of the test fungus compared to control (67 mm) and lowest mycelia growth (20.67 mm) in turmeric was recorded (Table   3).

Mycelia Growth of MoO in Vitro
Among those eight botanicals in 1:2 (w/v) at 7 DAI, highest mycelia growth was recorded in neem (29 mm) of the test fungus compared to control (46.67 mm) and lowest mycelia growth (12 mm) in turmeric was recorded (Table 4).  Among those eight botanicals in 1:2 (w/v) at 10 DAI, highest mycelia growth was recorded in neem (30 mm) of the test fungus compared to control (53.33 mm) and lowest mycelia growth (16 mm) in turmeric was recorded (Table 4).
Among those eight botanicals in 1:2 (w/v) at 14 DAI, highest mycelia growth was recorded in neem (32 mm) of the test fungus compared to control (67 mm) and lowest mycelia growth (18 mm) in turmeric was recorded (Table 4).

Mycelia Growth of MoOIn Vitro
Among those eight botanicals in 1:1 (w/v) at 7 DAI, highest mycelia growth was recorded in garlic (29 mm) of the test fungus compared to control (46.67 mm) and lowest mycelia growth (8.33 mm) in turmeric was recorded (Table 5).
Among those eight botanicals in 1:1 (w/v) at 10 DAI, highest mycelia growth was recorded in garlic (38 mm) of the test fungus compared to control (53.33 mm) and lowest mycelia growth (9 mm) in turmeric was recorded (Table 5).
Among those eight botanicals in 1:1 (w/v) at 14 DAI, highest mycelia growth was recorded in garlic (40 mm) of the test fungus compared to control (67 mm) and lowest mycelia growth (9 mm) in turmeric was recorded (Table 5).   Each value is the mean of three replications. In a column, figure having same letter(s) do not differ significantly at 5% level by DMRT.

Efficacy of Botanical Ethanol Extracts in Controlling Radial Mycelia Growth of MoO in Vitro
significantly different and superior to rest of the treatments ( Figure 5). The next botanicals in order of merit were garlic 1:1 (w/v) with 92.68% growth inhibition compared to control. The next botanicals in order of merit were garlic 1:2 (w/v), ginger 1:2 (w/v) and garlic 1:4 (w/v) with 89.02%, 87.80% and 85.37% mycelia growth inhibition respectively of the test fungus as compared to control. After that in order of merit were turmeric 1:1 (w/v) and ginger 1:1 (w/v) with 81.71% growth inhibition of the test fungus as compared to control. Turmeric 1:4 (w/v) with 25.61% growth inhibition was found to be the least effective botanicals in vitro. Graphical representation was shown in Figure 6.

2) In Vitro Mycelia Growth of MoO at 14 DAI
In vitro mycelia growth of MoO on different botanical treatment with ethanol extracts at 14 DAI was found significantly different (Table 6). Lowest mycelia growth was recorded in neem that is 7.33 mm, 6 mm and 4.67 mm at 1:4, 1:2 and 1:1 (w/v) respectively at 14 DAI. Maximum growth inhibition of MoO was achieved with neem in all of it's concentrations with 92.62%, 90.52% and 88.42% mycelia growth inhibition respectively which was significantly different and superior to rest of the treatments ( Figure 5). The next botanicals in order of merit was allamonda 1:1 (w/v) with 66.84%, garlic 1:2 (w/v) with 66.32% and ginger 1:1 (w/v) with 66.32% growth inhibition compared to control. The next botanicals in order of merit were ginger 1:2 (w/v) and garlic 1:4 (w/v) with 64.20% and 63.16% mycelia growth inhibition respectively of the test fungus as compared to control. After that in order of merit were aloe Vera 1:2 (w/v), turmeric 1:1 (w/v), allamonda 1:4 (w/v) and aloe Vera 1:1 (w/v) with 55.79%, 53.69%, 52.63% and 51.57% growth inhibition respectively of the test fungus as compared to control. Kalijira 1:2 (w/v) with 11.57% growth inhibition was found to be the least effective botanicals in vitro. Graphical representation was shown in Figure 6. [32] Recorded that A. indica leaf extract @ 0.5% was found most effective in minimizing the mycelia growth of both the pathogens28.35 mm and 27.12 mm, closely followed by P. glabra leaf extract 29.57 and 30.10 mmin the same concentration, 96 hrs after incubation in vitro. Our results are in accordance to those [33] who studied that among the botanicals the spraying of Achook, NeemAzal T/S, Neem gold and Tricure shows significant reduction in disease American Journal of Plant Sciences  severity against blast of rice, along with improving yield attributes, increasing the 100-grain weight and grain yield. [34] Also reported that neem extracts reduced the mycelia growth of M. oryzae. [25] Also had same opinion that water and leaf extracts/oil extracts of seeds of Azadirachta indica (neem) reduced radial growth of mycelium of M. grisea in vitro and the development and spread of blast disease in green house.

In Vitro Mycelia Growth of MoO at 7 and 14 Days after Inoculation at 1:4 (w/v) Botanical Water Extracts
Among those eight botanicals mixed with ethanol in 1:4 (w/v) at 7 DAI, highest mycelia growth was recorded in turmeric (40.67 mm) of the test fungus compared to control (54.67 mm) and lowest mycelia growth (0 mm) in neem and allamonda was recorded (Table 7).  Among those eight botanicals mixed with ethanol in 1:4 (w/v) at 14 DAI, highest mycelia growth was recorded in kalijira (53.33 mm) of the test fungus compared to control (63.33 mm) and lowest mycelia growth (7.33 mm) in neem was recorded (Table 7).
Highest %growth inhibition was recorded in neem and allamonda at 1:4 (w/v) ethanol extract. The observations supports the findings of [35] who found the efficacy of plant parts extract of neem seed kernel, neem oil, Pongamia spp. extracts Panchagavya and Asafoetida spp. extract in descending order against rice blast fungus. [36] Observed that extracts from C. Arabica, N. tabacum, A. vera, A. indica, were found significant to manage rice blast disease in vitro and in vivo.

In Vitro Mycelia Growth of MoO at 7 and 14 Days after Inoculation at 1:2 (w/v) Botanical Ethanol Extracts
Among those eight botanicals mixed with ethanol in 1:2 (w/v) at 7 DAI, highest mycelia growth was recorded in kalijira (36 mm) of the test fungus compared to control (54.67 mm) and lowest mycelia growth (0 mm) in neem and allamonda was recorded (Table 8).
Among those eight botanicals mixed with ethanol in 1:2 (w/v) at 14 DAI, highest mycelia growth was recorded in kalijira (56 mm) of the test fungus compared to control (63.33 mm) and lowest mycelia growth (6 mm) in neem was recorded (Table 8).  Each value is the mean of three replications. In a column, figure having same letter(s) do not differ significantly at 5% level by DMRT. Each value is the mean of three replications. In a column, figure having same letter(s) do not differ significantly at 5% level by DMRT.

In Vitro
to control (54.67 mm) and lowest mycelia growth (0 mm) in neem and allamonda was recorded (Table 9). Among those eight botanicals mixed with ethanol in 1:1 (w/v) at 14 DAI, highest mycelia growth was recorded in kalijira (48.67 mm) of the test fungus compared to control (63.33 mm) and lowest mycelia growth (4.67 mm) in neem was recorded (Table 9).

Conclusions
Studies were conducted to determine the effect of aqueous extracts of Kalijira (Nigella sativa), Turmeric (Curcuma longa), Ginger (Zingiber officinalis), Garlic (Allium sativum), Onion (Allium cepa), Neem (Azadirachta indica), Allamanda (Allamanda cathartica), Aloevera (Aloe vera) for control of rice blast pathogen Magnaporthe oryzae oryzae in vitro following poisoned food technique. Maximum growth inhibition of MoO was achieved with water extract of turmeric in 1:1 (w/v) with 86.57% mycelia growth inhibition which was significantly different and superior to rest of the treatments.
In case of botanicals extracted in ethanol, maximum growth inhibition of MoO was achieved with neem in all tested concentrations with 92.62%, 90.52% and 88.42% mycelia growth inhibition respectively which was significantly different and superior to rest of the treatments.
A field trial is suggested for testing the field performance of turmeric water extract and neem ethanol extract along with a chemical fungicide to control rice blast in field condition.