In vivo and In vitro Management of Purple Blotch of Onion by Using Fungicides and Plant Extracts

An investigation was carried on the management of purple blotch disease of onion (Alternaria porri) during 2015-2016 at BARI, Joydebpur, Gazipur, Bangladesh to see the effectiveness of fungicides in controlling the disease and evaluate the performance of some plant extracts in controlling purple blotch disease of onion seed crop. In in vivo, the effectiveness of 10 fungicides (Antracol, Folicur, Iindofil M-45, Metaril, Rovral, Score, Sulcox, Sun copperoxy, Unilax and Vonot 306) were screen against the disease. The fungicides were applied at 0.2%, three times at 10 days interval. Of them Rovral appeared the best followed by Score. Folicur, Antracol, Metaril, Sulcox and Indofil M-45. More or less similar trend of the fungicides became evident when they were tested against Alerternaria porri in in vitro. While fungitoxicity of seven plants extracts (Dhatura, Dholkolmi, Garlic, Ginger, Marigold, Neem and Nymbicidine) was tested in field condition. Nymbicidine showed significantly the best performance in reducing the disease incidence and giving higher yield.


Introduction
Onion (Allium cepa L.) is the most important one and familiar throughout the world and also used as popular vegetable among the spices crops. The major onion growing districts of Bangladesh are: Faridpur, Cumilla, Dhaka, Dinajpur, Jessore, Pabna and Rajshahi. It is an important vegetable in Australia, Belgium, India, Japan, United Kingdom, USA and some other countries. However, bulb onion is still the most popular here. The present production of onion is around 18, 67,000 MT from 4, 59, 000 acres of land [1],which is low compared to other onion growing countries of the world.
A number of diseases are associated with onion crops [2] - [5].Among them the well documented disease is onion purple blotch incited by Alternaria porri (Ellis) which is noted as a major disease throughout the world including Bangladesh [4]- [7].In India, purple blotch of onion is a major devastating and widespread disease and causes serious yield reduction [8].The disease is also a threat for seed production of onion [9]- [11].
Damage of foliage and breaking of floral stalks due to purple blotch resulting failure of seed production of onion are common [2]. [12]reported that the disease causes 41-44% yield loss in Bangladesh. [13]reported that under favorable environmental conditions complete failure of the crop takes place and there will be no seed setting. In India, the disease causes 20 to 25 per cent loss in seed yield [14].
Literatures from home and abroad indicate that many explored to find out suitable control methods of the disease, viz. resistant variety, date of planting, use of fertilizers, bulb size and chemical control with fungicides [16]- [23].Available literatures indicating quite a largenumber of plant species have been reported to have the antipathogenic activity [24]- [27].
In Bangladesh, no resistant source is available. The varieties grown in the country are highly susceptible to the disease. Role of environmental factors on disease development has not yet been studied systematically. Therefore, quite a little information is available on fungicidal control; and mostly those are on bulb production only [2], [10] but not on seed production. So, the present study was undertaken with the following objectives: 1) To screen the effectiveness of fungicides in controlling the disease and 2) To evaluate the performance of some plant extracts in controlling purple blotch disease of onion seed crop.
Intercultural operation: A total of three times irrigation was done at 30, 60 and 90 days after transplanting by flood method.The dead or sick seedlings were replaced by healthy seedlings within a week after plantation. The damaged plants were also replaced by border plants through gap filling.Weeding and mulching were done as and when required to keep the crop free from weeds, for better soil aeration and conserve soil moisture. Mechanical support was provided in each plot using bamboo stick to keep the plant erect and to protect them from the damage caused by storm and high winds.
Collection of data: Data were recorded on lesion area, disease severity, PDI (Percent disease index), stem height, number of stem per hill, umbel diameter, weight of seeds/umbel, weight of seeds per plot, yield per plot was converted into yield per hectare. Lesion area was expressed in millimeter by measuring length and breadth of the leaves. Disease severity of purple blotch was assessed using 0-5 scale [13], as follows, by selecting 10 plants randomly from each plot and used for PDI (percent disease index) estimation. 0 = no disease symptoms 1 = a few spots towards the tip, covering less than 10% leaf area 2 = several dark purplish brown patches covering less than 20% leaf area 3 = several patches with paler outer zone, covering up to 40% leaf area 4 = long streaks covering up to 75% leaf area or breaking of leaves/stems from the centre 5 = complete drying of the leaves/stems or breaking of the leaves/stems from the base.
The per cent disease index (PDI) was calculated by using formula given by [28], [29] as follows: Total sum of numerical ratings PDI = x 100 Number of observation x Maximum grading Umbel diameter was taken by randomly selecting 10 umbels from each replication. Weight of seeds/umbel was assessed by recording seed weight of 10 umbels per replication.The crop was harvested within 100 -125 days after transplanting.

Efficacy of some Fungicides in Controlling Purple Blotchof Onion
Seedlings of local cultivars were included under the study. The unit plot size was 2.0 x 2.0m, keeping 20cm and 10cm spacing respectively for row to row and plant to plant.Apparently healthy seedlings were transplanted on 20.12.15.

Treatments (Fungicides):
The experiment consisted of 11 treatments including control. The treatments were as follows: Application of fungicides: Spray of fungicides was initiated after the disease symptoms detected in the experimental plot. A total of three sprays were performed at an interval of 10 days. The experimentwas scheduled following RCBDhaving three replications.

Bioassay of Fungicides and their Concentrations in ControllingGrowth of Alternaria porri inin vitro
Collection and Isolation of the fungus Ideal purple blotch diseased samples of onion was collected from experimental field of BARI, Gazipur and brought in the Plant Pathology Central Laboratory. Diseased samples were processed into small pieces, sterilized with 10% chlorox solution for three minutes, rinsed with sterile water and finally placed on Petri plates (9 cm dia.) containing host extract media. The composition of the host extract Media were onion leaf (200g), dextrose (15g), agar (20g) and water (1000 ml). Onion leaf was cut into small pieces, put in 500 ml water andboiled for 30 minutes. Then supernatant extract was decanted. Within the supernatant other ingredients were added with rest quantity of water to make one litre. The medium was then autoclaved at 121 o C at15 PSI for 20 minutes. Aftersterilization the medium (at 40-45 o C) was poured at the rate of 20 ml per Petri plates. After solidifying of the media inoculated Petri plates were incubated at 251 o C for five days. The isolated fungus was identified by using the keys outlined by [30]. 2000, 1000, 500 and 250 ppm. Requisite quantities of fungicides were mixed in fixed quantity of distilled water to achieve desired concentrations. In case of control, only sterile water was used. Each concentration was replicated thrice.

Bioassay of Fungicides:
Poisoned food technique was followed [31]. Potato dextrose agar medium was used and poured at the rate of 15 ml per Petri plate (9 cm). Before pouring of medium one ml of fungicidal solution (for each concentration of fungicides) was added in each Petri plate. After adding medium the plates were shaken in swirling motion for uniform mixing. After solidifying of the media, the centre of each Petri plate was inoculated with a 3 mm mycelial disc, cut from the edge of 10 days old fresh culture of A. porri. The inoculated Petri plates were then incubated at 25  1 o C for 10 days.
Collection of data: After incubation the mycelia growth of the fungus from each concentration of the fungicide was recorded.

Design:
The experiment was arranged following Completely Randomised Design.

Effect of Plant Extracts in Controlling Purple Blotch Disease ofOnion Seed Crop
Treatments (Plant extracts): There were eight treatment including control. The treatments were as follows: Garlic Allium sativum T 4 Ginger Gingiberofficinale T 5 Marigold Tagetespatula  T 6 Neem Azadirachta indica T 7 Nymbicidine Azadirachta indica T 8 Control - From T 1 to T 6 , the dose of each extract was used at the rate of 15 ml per 1000 ml water. Treatment T 7 , Nymbicidine was applied at the rate of 0.2% per 1000 ml water.

Application of Plant Extracts:
A total of five sprays were scheduled at 10 days interval, initiating just after the onset of disease symptom in experimental field.
Analysis of data: Data were analysed statistically using MSTAT-C computer Program. Data were transformed, whenever necessary, following Arcsine transformation. Means of treatments were separated using Duncan's Multiple Range Test (DMRT) [32].

Efficacy of Some Fungicides in Controlling Purple Blotch Disease of Onion
There was significant variations among the fungicides in relation to disease parameters of purple blotch disease of onion and the corresponding effects on yield (Table 1 and 2). .03% more yields were obtained respectively (Fig. 2), by using Antracol, Folicur, Indofil, Metaril, Rovral, Score, Sulcox, Sun Copperoxy, Unilax and Vondozeb.

Bioassay of fungicides and their concentrations in controllinggrowth of Alternaria porri in vitro
Results on the effectiveness of fungicides and their concentrations on mycelial growth of A. porri are presented in Table 3. Irrespective of concentrations, Rovral and Score appeared the best in controlling growth of A. porri and they showed statistically similar effect. The fungicide Folicur   .000 a 9.000 a 9.000 a 9.000 a -Means bearing same letter within the same column do not differ significantly at 5% level following DMRT.

Effect of Plant Extracts in Controlling Purple Blotch Disease of Onion Seed Crop
Significant differences among the effects of plant extracts became evident in controlling purple blotch of onion as well as in the seed contributing characters (Tables 4 and 5). The maximum stem height was recorded in the crops where Dhatura (T 1 ) extract (62.09 cm) was applied followed by T 6 (Neem), T 7 (Nymbicidine), T 4 (Ginger), T 3 (Garlic), T 8 (Control), T 5 (Marigold) and T 2 (Dholkalmi). Statistical analysis revealed that significantly higher stem height was measured under the treatment T 1 and this was statistically similar to T 6 and Nymbicidine (T 7 ) but differed significantly with the rest of the treatments ( Table 4). The treatment T 2 gave the lowest stem height and the differences were statistically insignificant with T 3 , T 5 and T 8 . In case of stem number per hill, it ranged from 1.86-2.47, where the minimum and the maximum number of stem per hill were recorded in T 8 and T 1 , respectively. The effect of treatments on producing number of stem per hill under the treatments T 1 , T 2 , T 4 , T 5 , T 6 and T 7 were insignificant. Significantly fewer numberof stem per hill was recorded in T 8 which was statistically similar to those of T 2 and T 3 .
The differences on the effects of plant extracts on the lesionarea were also pronounced under the study and it ranged from 18.40-25.90mm 2 . In order of affectivity of plant extracts in reducing the lesion area of purple blotch, the treatments can be arranged as: T 7 (18.40 mm 2 ), T 6 (19.13 mm 2 ), T 3 (19.20 mm 2 ), T 1 (20.40 mm 2 ), T 2 (20.60 mm 2 ), T 4 (21.33 mm 2 ), T 8 (25.10 mm 2 ) and T 5 (25.90 mm 2 ). Significantly better response in minimizing the purple blotch lesion area was measured under the treatment T 7 which differed significantly with all the rest of the treatments. The effect of treatments noted at T 1 , T 2 , T 3 , T 4 and T 6 showed statistically similar in reducing lesion area and they showed significantly lower lesion area compared to those of T 5 and T 8 . Regarding PDI value, the minimum PDI was computed at T 7 (37.66%). The treatment, T 1 ; ranked next to T 7 followed by T 6 (43.66%); T 2 (45.66%), T 4 (52.00%), T 3 (64.00); T 5 (64.00%) and T 8 (86.33%). Statistical analysis revealed that, among the plant extracts, Nymbicidine (T 7 ) was the best, which significantly reduced the PDI of purple blotch compared to the others ones except T 1 . The differences between the reductions of PDI under the treatments T 1 and T 6 was insignificant and the treatments gave significantly lower PDI compared to T 2 , T 3 , T 4 , T 5 and T 8 (Fig. 3). The treatments T 2 , T 4 and T 6 showed statistically similar performance on reduction of PDI. Treatment T 8 has shown significantly higher PDI and it differed significantly with the rest of the treatments. The maximum PDI reduced over control was shown by T 7 (56.37%) followed by T 6 (51.20%), T 1 (49.84%), T 2 (47.10%), T 4 (39.76%) and T 3 and T 5 (30.49%).
The treatments showed a wide variation on yield contributing parameters both numerically and statistically. The influence of treatments (Plant extracts) on umbel diameters ranged from 46.23-53.83 mm. The maximum umbel diameter was recorded in T 7 followed by T 6 (52.46 mm), T 1 (51.69 mm), T 4 (50.55 mm), T 5 (49.67mm), T 3 (48.67 mm), T 2 (48.43 mm) and T 8 (46.23 mm). The umbel diameter recorded by T 7 differed significantly with those of other treatments (Table5). However, there was no significant variation between the treatments, T 1 and T 6 on umbel diameter; so were T 4 and T 5 . Significantly lowerumbel diameter was recorded in T 8 and it differed significantly with the rest of the treatments. In case of weight of seed per umbel, significantly higher amount of seed was harvested from the umbels of T 6 treated plots but statistically it showed similar effects with all the rest of the treatments except T 8, where no plant extract was sprayed. Though significantly lower seed weight per umbel was assessed in T 8, it was statistically similar to those of T 2 , T 3 , T 4 , T 5 and T 7 .
The yield per plot ranged from 55.00-135.00g with the minimum and the maximum given by T 8 and T 7 , respectively. The treatment T 6 gave the second highest per plot seed yield (109.30 g) followed by T 1 (92.67g), T 5 (82.67g), T 4 (72.00g), T 3 (66.00g) and T 2 (65.00g) ( Table 5). Statistical analysis indicated that significantly higher seed yield was harvested under the treatment T 7 and it differed significantly with all other treatments. The treatment T 6 gave significantly lower seed yield than T 7, but gave significantly higher per plot seed yield over T 1 , T 2 , T 3 , T 4 , T 5 and T 8 . The effect of T 1 and T 5 on per plot seed yield was insignificant. Again, the effect of T 2 , T 3 and T 4 on same parameters was insignificant among themselves. About 68. 49, 18.18, 20.00, 30.90, 50.30, 98.72 and 145.45% yield increased over control was estimated respectively, by those of T 1 , T 2 , T 3 , T 4 , T 5 , T 6 and T 7 treatments (Fig.4).   [44].According to [45] more than 75% disease reduction was obtained using Rovral. [46]opined that iprodione (Rovral) performed excellent in reducing disease intensity of purple blotch and gave highest yield. The effectiveness of Folicur, Metaril, Antracol against purple blotch have also been suggested by [22], [23], [40], [43] and [47]- [53].Under the study Indofil M-45 (mancozeb) was found not so effective to control A. porri incidence compared to Rovral, Score, Folicur and Metaril. On the contrary good control of A. porri by Dithane M-45 (Mancozeb) have also been reported by many workers under both in vitro and in vivo [2], [8]- [11], [20], [23], [25], [39], [50] and [54]- [60]. [9], [13], [58] and [61]stated that Dithane M-45 was the best among the fungicides to reduce the purple blotch disease incidence and maximizing bulb yields. [62]reported that mancozeb reduced the purple blotch of onion by 23.6% and increased the yield by 35.0%. The potentiality of copper fungicides against A. porri has been documented by many researchers [10], [22], [23], [43], [50], [59]- [61] and [63]- [65]which differed with the present investigation where coper fungicides like sun Copperoxy and Sulcox appeared to be less effective to A. porri. According to [8], however, copperoxycloride was the best to reduce purple blotch and to maximize the highest bulb yield; which the results of this information fail to support. It may indicate the need to investigate wheatherA. porri has developed resistance against copper based fungicides, in the meantime.
Results on evaluation of fungi toxicity of plant extracts to control purple blotch of onion indicate that all of them have some potentiality against the disease but it varied widely among the extracts of plant origin. Of them Nymbicidine, Neem leaf extracts and Datura performed better compared to rest of the plant extracts. Dholkolmi plant extract shows less effectiveness against purple blotch (A. porri); among the plant extracts. The fungitoxic potential of Neem and Datura have been supported by [26], [27]

Conclusions
In a field study fungitoxicity of 10 fungicides were tested to locate effective one(s) to combat purple blotch (A. porri) disease of onion using seedlings of local cultivars, transplanted in unit plot of 2.0 x 1.2 m. The fungicides employed were: Antracol, Folicur, Indofil M-45, Metaril, Rovral, Score, Sulcox, Sun Copperoxy, Unilax and Vondozeb. All the fungicides were used at the rate of 0.2%. A total of three sprays were executed at 10 days interval. It appeared that there were significant variations in effectiveness among the tested fungicides against A. porri. Among them Rovral gave best control of the disease (lesion area and PDI) followed by score, Folicur, Antracol, Metaril,Sulcox, Indofil M-45, Sun Copperoxy, Unilax and Vondozeb. More or less similar trend were shown by fungicides in yield contributing characters.
The effectiveness of the fungicides tested in vivo was also evaluated in vitro to evaluate their performance against A. porri using poison food technique. The fungicides were tested at four concentrations viz. 250, 500, 1000 and 2000 ppm. Irrespective of concentrations Rovral and Score appeared the best one in inhibiting the growth of A. porri followed by Folicur. Among the fungicides Vondozeb and Unilax were proved ineffective to restrict the growth of A. porri.
The antifungal effect of six plant extracts (Datura, Dholkalmi, Garlic, Ginger, Marigold and Neem) and one commercial plant product, namely Nymbicidine (ACI crop care division) was evaluated under field condition. Under the study, bulbs of one of local cultivar was planted in unit plot size of 3.0 x 1.2 m. Nymbicidene was applied at the rate of 2 ml/litre of water while 15 ml/litre of water each plant extract was applied to best their efficiency. Each treatment was replicated thrice. As the crop was seed crop, plant extracts was applied five times at an interval of 10 days. It was found that all the plant extract had some degree of potentiality in reducing disease contributing characters. Of them Nymbicidine T 7 performed best in minimizing the purple blotch and it differed significantly from rest of the plant extracts. This was followed by T 1 (Datura), T 6 (Neem), T 2 (Dholkolim), T 4 (Ginger), T 3 (Garlic) and T 5 (marigold). Nymbicidine also gave the highest seed yield.
Based on the findings of the present investigation the following conclusions may be made. i) Fungicides like Rovral, Score, Folicur, Metaril are highly effective to check purple blotch disease of onion ii) Nymbicidine, Datura, Neem and Dholkolmi are potential plant extracts against Alternaria porri.