CONTROL OF Bipolaris oryzae (Breda de Haan) USING Curcuma longa (Linnaeus) EXTRACT AND EFFECT OF THIS EXTRACT ON RICE SEED PHYSIOLOGY 1

– This study was conducted to evaluate the use of Curcuma longa (Linnaeus) extract in the in vitro control of Bipolaris oryzae (Breda de Haan) and to characterize the effect of this extract on rice seed germination. A completely randomized arranged in a factorial experimental design was used: three isolates of B. oryzae from rice seed from different rice-growing regions of Rio Grande do Sul (Fronteira Oeste, Campanha, and Sul) were tested with three concentrations (20, 40, and 80 mg/mL) of C. longa plus a control treatment (0 mg/mL). Each reaction was repeated in quadruplicate. The effect of the extract upon the disease development was evaluated based on mycelial growth (PMG) and spore production; rice seed germination was evaluated using a germination test (Germitest®). The PMG results demonstrate that the treatments were effective in reducing PMG, with a stronger response observed as the concentration of the extract increased. An average inhibition of 84% of sporulation was observed for the tested strains compared with the control treatment. There were, however, no significant differences in terms of seed germination test with the different C. longa concentrations. Therefore, treatment of rice seeds with C. longa extract does not affect seed germination but positively inhibits mycelial growth and sporulation, affecting the in vitro sporulation of the different isolates of B. oryzae

The symptoms of brown spot can be observed in several parts of the plant, but it has the greatest effect when present on the leaves, where it reduces the active photosynthetic area, or on the grains, where it affects their quality, and can cause yield losses of up to 72% (MALAVOLTA et al., 2007).Currently, control of this disease relies on the use of chemicals, as there are few cultivars available with resistance to the pathogen (GROHS et al., 2010).As reported by SOSBAI (2016), fungicides of the triazol group or mixtures of triazols and strobilurins have been reported to be effective for the control of B. oryzae.Unfortunately, these agrochemicals increase the production costs of the crop and can have negative impacts on the environment (KAGALE et al., 2004;HARISH et al., 2007).
The use of alternative methods of disease control is becoming more popular to help minimize the negative effects of agrochemicals and facilitate sustainable food production.Many of these methods rely on the use of natural plant extracts and oils that are sources of a wide spectrum of compounds with antimicrobial action that can be utilized in integrated disease management (GURJAR et al., 2012).For example, Cymbopogon citratus and Thymus vulgaris essential oils have been shown to be effective seed treatments for the control of rice seed-spotting fungi (NGUEFACK et al., 2008).
Turmeric (Curcuma longa Linnaeus) is known for its antibacterial and antioxidant pharmacological functions (ARAÚJO; LEON, 2001).The use of C. longa root extract proved effective in the in vitro control of phytopathogens such as Colletotrichum gloeosporioides and Fusarium oxysporum Schlecht.(SINGH;SINGH;MAURYA, 2002).When used in vivo, it reduced the severity of early blight (Alternaria solani) in tomato plants under greenhouse conditions (BALBI-PEÑA et al., 2006).
Although Nguefack et al. (2008) found that the natural products of C. citratus and T. vulgaris were effective in the control of rice grain-affecting fungi, little is known about the effect of those extracts upon the crop.Some volatile compounds produced as plant secondary metabolites are known to have allelopathic or even phytotoxic effects on seeds, preventing them from germinating (ISMAN, 2006).The treatment of corn seeds with different essential oils resulted in a drastic reduction in seed germination, indicating that some oils that are effective treatments against storage fungi may also be negatively affecting seed physiology (BRITO et al., 2012).We, therefore, aimed to evaluate the use of C. longa extracts for in vitro control of B. oryzae and characterize its effect upon the germination of rice seeds.
The effect of C. longa on pathogen development was evaluated through mycelial growth and spore production; the effect of the extract on the rice seed germination was also investigated.
The experimental design was completely randomized and was arranged factorially with the three isolates tested against three extract concentrations (20, 40, and 80 mg/mL) plus one control treatment (0 mg/mL).Four replicates were included for each treatment.
To evaluate both mycelial growth and sporulation, the fungus was grown on potato dextrose agar (PDA) culture media (Acumedia®) modified with broth containing different concentrations of C. longa to achieve final C. longa extract concentrations of 0, 20, 40, and 80 mg/mL PDA.The media with extracts were autoclaved and poured into Petri dishes (80 × 15 mm).In the central region of each dish a 5-mm-diameter disk containing fungal mycelium was added; the plates were incubated in a growth chamber at 25°C with a 12-hour light photoperiod.
Mycelial growth was evaluated daily by measuring the diameter of the colonies with a digital pachymeter (Digital Vernier Caliper®); measurements were taken 24 h after the mycelium disk was placed in the culture medium and continued until the colony in one of the treatments spanned the total diameter of the Petri dish.
The mean mycelial growth of each colony (CMC) and the mycelia growth index (IVCM) were evaluated daily for the first 5 d after inoculation using the formula , where IVCM = mycelial growth index; D = current average diameter of the colony; Da = average diameter of the colony on the previous day; N = number of d after To evaluate spore production, the number of spores produced per treatment was determined 10 days after inoculation.The spore suspension was obtained by collecting 10 disks mycelium for each replicate; these were transferred to test tubes containing 10 mL sterile distilled water and were then vortexed (PHOENIX ® , AP 56) before the number of spores was counted using a Neubauer chamber.The results were analyzed through regression analysis with a decreasing exponential equation, determined using the default parameters in the SigmaPlot 10.0 program.
The effect of C. longa extracts upon seed germination was determined using Germitest® paper towels that were sterilized and moistened with sterile distilled water containing different concentrations of powdered C. longa extract (0, 20, 40, and 80 mg/mL) according to the previously described seed analysis method (BRASIL, 2009).Before treatment, the seeds were analyzed for the presence of fungi, particularly B. oryzae, using the previously described blotter test (BRASIL, 2009).As a high incidence of storage fungi (35%) was detected, the surface of the seeds was sterilized by washing with 1% sodium hypochlorite for 2 min followed by three washes with sterile distilled water.
For each treatment, 200 seeds of the IRGA 424 cultivar were divided into four replicates of 50 seeds each.The seeds were soaked for 10 min in sterile water containing different concentrations of C. longa extract before being germinated in an incubation chamber in the vertical position at 25°C with 70% relative humidity and a 12-h photoperiod.
Germination rates were evaluated after 14 d when the percentage of germinated seeds, damaged abnormal seedlings, infected abnormal seedlings, hard seeds, and dead seeds were estimated as previously described by BRASIL (2009).Next, ten seedlings were randomly selected for each replicate from which measurements of both the shoot and the root length were taken using a graduated ruler, obtaining the average length for each in cm.The weight of green plant mass was established using a precision balance The data in percentages were transformed into arcsine (SANTANA; RANAL, 2004) and submitted to analysis of variance.The means were compared using the Tukey test at a 5% probability threshold using Assistat 7.6 software.

RESULTS AND DISCUSSION
Mycelial growth (CMC) was evaluated until the fifth day, at which point the mycelium in the controls had reached the edge of the plates.Figure 1 shows that treatment with C. longa extract was effective in reducing CMC, with greater extract concentrations resulting in reduced CMC.After 5 d of growth, a reduction in CMC of 40% to 60% was observed for the three isolates at the highest concentration of C. longa extract (80 mg/mL).These levels of mycelial growth inhibition were similar to the levels of inhibition observed for F. verticillioides when exposed 20 μg/mL of turmeric extract, where mycelial growth of colonies was reduced by 56% (AVANÇO et al., 2017).
The IVCM was also most affected by the highest concentration of C. longa extract, with a similar 40-60% reduction observed for all three isolates treated with 80 mg/mL C. longa extract (Figure 2).C. longa extract also affected sporulation, with sporulation in the three isolates decreasing as the extract concentration increased.When treated with 80 mg/mL C. longa extract there was an 82.67%, 84.17%, and 83.34% reduction in sporulation for isolates 1, 2, and 3, respectively, when compared to the control treatment (Figure 3).This suggests that C. longa extract has properties that reduce in vitro sporulation of B. oryzae at the concentrations tested.(BALBI-PEÑA et al., 2006).Consistent with our results, these authors also showed a reduction in the spore production of B. oryzae exposed to different concentrations of C. longa.The mechanism by which this extract controls pathogen development may be related to compounds present in the rootstock of C. longa; these include curcuminoid phenolics, also known as curcumin, that are fungitoxic to phytopathogens (BALASUBRAMANYAM et al., 2003).Using C. longa oil for the control of Cyrtomium falcatum and F. moniliforme, Singh, Singh and Maurya (2002) observed complete inhibition of mycelial growth when a dose of 100 mg/L was used.
In this study, we have shown that different isolates of the same pathogen may have different responses to the presence of C. longa extract.While the C. longa treatments were effective against all three isolates, each isolate responded differently in terms of the evaluated variables.B. oryzae has a large number of nuclei in the cells of both the hyphae and the conidia (MCDONALD; LINDE, 2002;SOMAYEH et al., 2015), resulting in variations between isolates in terms of growth, sporulation capacity, colony coloration, and aggressiveness (SOUZA; RIBEIRO; GALLI, 1984; MOTLAGH; ANVARI, 2010).These differences were apparent in studies carried out in the 1980s and this high genetic variability may be associated with the inter-isolate differences observed in this study.In studies on the genetic resistance of 49 rice cultivars to eight monosporic isolates of B. oryzae, different plant responses to isolates of the same fungus were observed (MALAVOLTA; SOUZA; AMARAL, 1992).In addition to the intrinsic variability of the pathogen, the fact that the isolates used in this study were from different regions with edaphoclimatic characteristics may also have played a role.For example, work conducted by Valim-Labres et al. (1997) with isolates of B. sorokiniana from different regions showed that the virulence of the isolates was related to the genetic characteristics of the pathogen, the host genotype, and also to environmental pressure associated with geographic differences such as temperature, air relative humidity, and photoperiod.Taken together, these results reinforce the need for studies that elucidate and effectively demonstrate the factors intrinsic to the pathogen and its interaction with the environment and host that actually influence the virulence of the isolates.
We also examined the germination rates of seeds treated with C. longa extracts to determine whether the different doses used to treat the seeds had phytotoxic effects.There were no significant differences between the different concentrations of C. longa across any of the nine variables evaluated (Table 1).---------------------------%--------------------------------cm----- From the results (Table 1), seed germination rates were low in all treatments.There was, however, a high incidence of hard seeds that were likely dormant, and a high incidence of infected abnormal seedlings.This suggests that, despite the sterilization of the seeds, many may not have been contaminated with the pathogen under study, B. oryzae, but were instead contaminated with other storage fungi like Aspergillus sp. that can cause seedling abnormality.Lazzari (1997), cited in Guimarães et al. (2010), stresses that the fungal contamination by Aspergillus sp.is, in addition to its negative impact on the nutritional value of rice and public health, also responsible for yield losses.
Notably, no C. longa treatment at any dose tested resulted in any deleterious physiological effect, relative to the control (Table 1).Given that the main form of dissemination of the fungus is through seeds, this suggests that the treatment of seeds with C. longa extracts may be a promising method of controlling the B. oryzae pathogen.
Previous works examining the use of extracts and oils for the control of pathogens focused only on the pathogen and not on the effect of these treatments on the plant itself.Given that many plant extracts are known to trigger phytotoxicity in the plant, as was shown by the work of Brito et al. ( 2012) that identified a reduction in the germination of maize seeds treated with citronella and eucalyptus essential oils, this is an area of significant interest.
The work presented here is, therefore, a fairly novel resource where both the effect of the extract on the pathogen but also on the seed has been evaluated; this work will provide a basis for further studies examining the treatment of rice seeds with C. longa extract to control B. oryzae.

CONCLUSIONS
Extracts of C. longa effectively reduced mycelial growth of B. oryzae isolates at a range of concentrations, assessed through reductions in the mycelial growth index.This extract also inhibits spore production of B. oryzae isolates without interfering with the germination of rice seeds previously soaked in the extract.

Figure 3 .
Figure 3. Spore production or sporulation (×10 4 spores) of different isolates of Bipolaris oryzae on different concentrations (0, 20, 40, and 80 mg/mL) of Curcuma longa extract in potato dextrose agar, 10 days after mycelium in the control dishes had reached the edge of the dish.• isolate 1, ▲ isolate 2 and ■ isolate 3. Similar results were observed in the control of A. solani, where C. longa extracts at a concentration of 20% inhibited 78.6% of fungal sporulation (BALBI-PEÑA et al., 2006).Consistent with our results, these authors also showed a reduction in the spore production of B. oryzae exposed to different concentrations of C. longa.The mechanism by which this extract controls pathogen development may be related to compounds present in the rootstock of C. longa; these include curcuminoid phenolics, also known as curcumin, that are fungitoxic to phytopathogens (BALASUBRAMANYAM et al., 2003).Using C. longa oil for the control of Cyrtomium falcatum andF.moniliforme, Singh, Singh and Maurya (2002)
Means followed by the same letter in each column are not statistically different according to the Tukey test (P ≤ 0.05).