Management of Purple Blotch Complex of Onion in Indian Punjab

Six systemic fungicides viz., Kitazin 48 EC (iprobenfos), Tilt 25 EC (propiconazole), Folicur 25 EC (tebuconazole), Score 25 EC (difenoconazole), Amistar Top 325 SC (azoxystrobin 18.2% + difenoconazole 11.4%) and Nativo 75 WG (trifloxystrobin 25% + tebuconazole 50%), and two non-systemic fungicides viz., Indofil M-45 75 WP (mancozeb) and Kocide 77 WP (copper hydroxide), were evaluated under in vitro and field conditions for their efficacy to manage purple blotch complex of onion caused by Alternaria porri and Stemphylium vesicarium. Field efficacy of the fungicides at different concentrations were determined in controlling the purple blotch complex of onion under artificial epiphytotic conditions on bulb and seed crop (cultivar PRO-6) during the Rabi season 2014-2015 and 2015-2016, respectively. The triazole fungicides, tebuconazole and difenoconazole proved superior in inhibiting growth of A. porri and S. vesicarium under in vitro conditions, respectively. Further, foliar sprays (3 for bulb crop and 4 for seed crop) of tebuconazole 25 EC (Folicur) @ 0.1 per cent at fortnightly interval most effectively managed purple blotch complex of onion under field conditions with highest Benefit: Cost ratio (8.75:1 and 88.7:1) in bulb and seed crop, respectively. Seed-to-seed method of onion seed production recorded significantly lower disease severity and higher seed yield than that of bulb-to-seed method under natural epiphytotic conditions. The present findings can be instrumental in devising strategy for the integrated management of A. porri, S. vesicarium singly as well as in complex, serious limiting biotic factors in onion production.


Introduction
Onion (Allium cepa L., 2n=16), a bulbous, biennial herb is the most important and one of the five most important fresh market vegetables worldwide (Cramer, 2000).Among vegetables, onion often called as "queen of kitchen" is one of the oldest known and an important crop grown in India.It contains the lachrymatory principle, a strong antibiotic, India is a traditional producer and assumes second global position in onion production with 19.40 million tonnes from 1.20 million hectares area (Anonymous, 2015).It is cultivated round the year throughout the country.Onion is highly susceptible to many foliar, bulb and root pathogens, which reduce its yield and quality (Cramer, 2000).Among the diseases, purple leaf blotch (PLB) caused by Alternaria porri (Ellis) Cif. and Stemphylium leaf blight (SLB) caused by Stemphylium vesicarium (Wallr.)Simmons, are the major diseases of onion world-wide affecting the foliage severely resulting in crop loss ranging from 30 to 100 per cent both in seed and bulb crop from year to year (Awad et al., 1978;Everts and Lacy, 1990a, b;Brar et al., 1990;Aveling et al., 1993Aveling et al., , 1994;;Chaput, 1995;Cramer, 2000) and are more prevalent in warm and humid environment (Suheri and Price, 2000a, b).
The low productivity of onion in India is chiefly attributed to prevalence of PLB and SLB in almost all the onion cultivated areas of Northern and Eastern regions (Gupta et al., 1996;Suhag and Bhatia, 2006).Although the PLB and SLB are common on both bulb as well as seed crop of onion, yet it is more damaging particularly in latter, every year in Punjab.The attack is common during March-April coinciding with the bolting of the crop and it may cause 35 to 100 per cent losses (Sharma, 1986).Being weather preferences similar for both PLB and SLB, they mostly occur synchronously on the same umbel bearing stalk resulting additive loss.The typical oval to elliptical lesions were colonised by either Alternaria porri, Stemphylium vesicarium or mixtures of both pathogens (Suheri and Price, 2001).The lesions containing A. porri alone or S. vesicarium alone or mixture of both were generally impossible to be differentiated from each other and thus, it was considered to be a complex problem (Suheri and Price 2000a, b).Uddin et al. (2006) also reported that the SLB pathogen (S. vesicarium) is first to initiate infection, which is followed by subsequent infection by the pathogen of PLB (A. porri) and hence, the disease is designated as purple blotch complex (PBC).Resistant cultivars, if available, are the most effective, practical, eco-friendly and economical choice.However, at present, no such resistant varieties are available in India (Shashikanth et al., 2007).Several workers have investigated the potential of conventional as well as new fungicides in different countries of the world for the management of PLB and SLB of onion, individually.However, a perusal of literature revealed that little work appears to have been done on PBC in Punjab and elsewhere.In view of economic importance of the crop, effect of the disease complex on yield and quality and perpetual occurrence of the disease complex , the present study was undertaken to determine in vivo and in vitro efficacy of novel fungicides and to evaluate the methods of seed production in order to standardize innovative onion purple blotch complex management approaches.

Isolation and Purification of the Pathogen Cultures
Isolations of the pathogens were made from the diseased leaf tissue collected from naturally infected onion fields.Typical diseased spot on the leaves were selected and cut into bits of about 1 to 1.5 mm with the help of sterilized scalpel, washed with sterilized distilled water and disinfected with 0.1 percent mercuric chloride (HgCl2) solution for 30 to 60 seconds.These disinfected bits were immediately rinsed in double sterilized distilled water repeatedly to remove the traces of mercuric chloride and toweled on sterilized filter paper, prior to their being aseptically transferred to Petri plates containing 20 ml of autoclaved potato dextrose agar (PDA) in a laminar flow and incubated at 25±1ºC in BOD incubator for 10 days.The resulting fungal culture was purified by hyphal tip technique in PDA slant both for A. porri and S. vesicarium.The culture of each fungus was further purified by single spore isolation technique.

Cultural and Morphological Studies of the Pathogens
The pathogens isolated in pure form were sub cultured in Petri plates (90 mm diameter) on potato dextrose agar and incubated at 25±1ºC.The observations on colony appearance and growth of the pathogens were recorded for 10 consecutive days.
The ten days old culture of each fungus was used to for morphological characterization and confirmation of identity of isolated pathogens.Identification of the each fungus under study was made after examining 100 conidia under compound microscope (40x).Stage and ocular micrometer were used to measure the length, breadth, beak length and number of septa of the fungus after the calibration of the microscope.The average length and breadth of the conidial body, beak and number of septa were recorded.These observations were compared with those of the standard measurements given by Ellis (1971) and Simmons (1969) to identify the pathogens.
All the systemic fungicides were tested at a series of concentrations viz., 0.5, 1, 10, 25, 50, 100 and 200 µg/ml, respectively while the non-systemic fungicides were tested at a series of concentrations viz., 0.5, 1, 10, 25, 50, 100, 200, 500 and 1000 µg/ml, respectively.The stock solution of the chemicals prepared in sterilized distilled water was added under aseptic conditions in 250 ml flask containing potato dextrose agar medium so as to have the final concentrations (on a.i.basis) of 0.5, 1, 10, 25, 50, 100, 200, 500 and 1000 µg/ml.The fungicide amended medium was poured in sterilized Petri plates, allowed to cool and seeded in the centre with 5 mm mycelial disc of actively growing culture of A. porri and S. vesicarium separately.The Petri plates containing un-amended PDA medium served as check.Each treatment was replicated four times using one Petri plate in each replicate under completely randomized design (CRD).The inoculated Petri plates were wrapped with parafilm to minimize the chances of contamination and incubated at 25±1ºC in BOD incubator.The observations on colony diameter of each fungus were recorded after the Petri plates (90 mm diameter) in control were completely filled with the mycelial growth of the respective fungus.The per cent inhibition in mycelial growth was worked out by using the formula devised by Vincent (1947).The efficacy of the fungicides was expressed in terms of ED50 and ED90 values calculated by probit regression analysis.The per cent inhibition at each concentration was transformed to the empirical probit value in point scale as given by Finney (1977).Then, the empirical probit values were regressed with log concentrations using simple linear regression.The constant parameters "a" and "b" were obtained.The ED50 and ED90 values were calculated using following formula:

In-Vivo Evaluation of Different Fungicides against Onion Purple Blotch Complex in Bulb and Seed Crop
Field efficacy of six systemic fungicides viz., Kitazin 48 EC (iprobenfos), Tilt 25 EC (propiconazole), Folicur 25 EC (tebuconazole), Score 25 EC (difenoconazole) @ 0.1 per cent, Amistar Top 325 SC (azoxystrobin 18.2% + difenoconazole 11.4%) and Nativo 75 WG (trifloxystrobin 25% + tebuconazole 50%) each @ 0.05 per cent, and two non-systemic fungicides viz., Indofil M-45 75 WP (mancozeb) @ 0.3 per cent and Kocide 77 WP (copper hydroxide) @ 0.25 per cent, were determined in controlling the onion PBC under artificial epiphytotic conditions on bulb and seed crop (cv.PRO-6) during the Rabi season 2014-2015 and 2015-2016, respectively.The nursery was raised during last week of October and transplanted in last week of December for bulb crop while for seed crop, the nursery was raised during last week of August and transplanted in last week of October.The bulb as well as seed crop was raised in the plot size of 2 x 2 m 2 at a spacing of 15 cm between rows and 10 cm between plants.The field trials were conducted in a randomized block design (RBD) with three replications along with the unsprayed check in the field area, Department of Plant Pathology as per standard agronomic practices, Punjab Agricultural University, Ludhiana (Anonymous 2013).Artificial epiphytotic conditions were created in the field by spraying mixed inoculum (conidial and mycelial bits suspension) of A. porri (1 x 10 4 propagules/ml) and S. vesicarium (1 x 10 4 propagules /ml) during evening hours in the first and last week of February.Foliar application of fungicides was started as soon as first symptom of the disease was noticed followed by two and three spray at 15 days interval in bulb and seed crop, respectively.
Twenty-five plants from each plot were randomly tagged for disease assessment on the 0 to 5 rating scale given by Sharma (1986)  The observations on disease severity were recorded 10 days after last fungicidal spray and the per cent disease index (PDI) was computed as per the formulae given by Wheeler (1969).
where, ∑SNR is the sum of numerical ratings N is the number of observations MDR is the maximum disease rating based on 0-5 scale The percent disease control was also calculated.The data on fresh bulb size (diameter in cm) were recorded on 25 randomly selected plants per plot with the help of digital Vernier caliper while the data on bulb and seed yield (kg or g per plot) were recorded after harvesting.Fresh bulb and seed yield per hectare was extrapolated in t/ha and q/ha, respectively.The economics of treatments was worked out by considering the prevailing rates of inputs, produce, labour charges and expressed as an incremental Benefit: Cost ratio.The B:C ratio was calculated using formulae Benefit: Cost (B: C) ratio

=
Additional benefits of the produce from each treatment Additional cost of each treatment

Evaluation of Method of Seed Production against Purple Blotch Complex in Onion
In order to determine the best method of onion seed production with the respect to incidence and severity of PBC, two recommended seed production method viz., Bulbto-seed and seed-to-seed, on two onion cultivars Punjab Naroya and PRO-6 were tested under natural epiphytotic conditions.The experiment was carried out in Plant pathology Farm, Punjab Agricultural University, Ludhiana during Rabi season 2015-2016 in randomized block design with four replications.The plot size of 2 x 2 m 2 was determined for all the replications.The description of each treatment used during the study is given as below: (i) Seed-to-seed method: In this method, healthy onion seeds were sown in nursery beds during last week of August 2015 and forty days old seedlings were transplanted in last week of October 2015 at a spacing of 15 cm between rows and 10 cm between plants.
For nursery raising, seeds were sown in 20 cm high nursery beds (size 2 x 1.5 m 2 ) under green house conditions by using 4-5 kg seeds per 200 m 2 for transplanting in an acre as per the Package of Practices for Vegetable Crops, Punjab Agricultural University, Ludhiana (Anonymous 2013).
(ii) Bulb-to-seed method: In this method, onion seeds were raised from the healthy bulbs produced during the previous year.The bulbs were planted at 60 cm x 45 cm in the first week of November.The required amount of bulbs was worked out and planted (plot size 2 x 2 m2 ).The plots were periodically observed for the number of infected plants and the intensity of PBC and disease incidence and per cent disease index (PDI) was computed according to the formula described in Section 3.1 of this Chapter.The data on the disease severity was recorded on 25 plants selected at random in the field following 0-5 rating scale given by Sharma (1986).The data on seed yield of each plot was also recorded in grams and the seed yield per hectare was extrapolated as q/ha.
The plots were periodically observed for the number of infected plants and the intensity of PBC and disease incidence and per cent disease index (PDI) was computed according to the formula described in Section 3.1 of this Chapter.The data on the disease severity was recorded on 25 plants selected at random in the field following 0-5 rating scale given by Sharma (1986).The data on seed yield of each plot was also recorded in grams and the seed yield per hectare was extrapolated as q/ha.

Statistical Analysis
The data of laboratory studies and field experiments were subjected to analysis of variance as per completely randomized design (CRD) and randomized block design (RBD) using statistical analysis software R Studio and the significance of differences between the treatment means were compared using Least Significant Difference (LSD) and Tukey's honest significant difference (HSD) test at 5 per cent level for proper interpretation of results.The percentages used in different experiments were subjected to arcsine transformations.The data on bulb and seed yield were extrapolated and expressed in t/ha and q/ha, respectively.

Cultural Studies
The colony growth of A. porri appeared creamy white after 3 days of incubation, which eventually changed to greenish grey and finally turned light olivaceous with distinct light green zonations.The colony was found slightly fluffy with light green raised margins in uniform concentric fashion with radial diameter of 87.43 mm on 10 th day of incubation period.Shahnaz et al (2013) studied the cultural characteristics of different isolates of A. porri and recorded that most of the isolates had fluffy growth on potato dextrose agar with colony colour varying from pinkish white through dull orange to olivaceous and black with distinct to diffuse patterns of zonation.
The colony growth of S. vesicarium appeared light grey to dirty white after 3 days of incubation, which finally turned greenish brown to brownish grey.The colony was found fluffy, umbonate to raised with filiform margin and appreciable amount of aerial mycelium with radial diameter of 60.95 mm after 10 days of incubation.Chowdhury (2013) reported that the colony colour of S. vesicarium varied from greenish brown to dirty white, deep grey to whitish, light grey to whitish, deep greenish white, light grey and dirty white to greenish with elevation varying from umbonate, raised to flat and texture varying from cottony, fluffy to velvety.Arzanlou et al (2012) reported that the colonies of S. vesicarium on potato carrot agar medium were grey to brownish grey, flat, attaining a diameter of 50 mm after 7 days with sparse aerial mycelium.

Morphological Studies
The microscopic study of A. porri revealed that mycelium of the fungus was initially hyaline which later became pale brown to olivaceous brown or smoky blended with purplish tinge.

In Vitro Evaluation of Different Fungicides against Alternaria porri
The data presented in   2).Iprobenfos 48 EC (Kitazin) was found to be least effective at lower concentrations.At higher concentration (200 µg/ml) it, however, recorded 62.79 per cent colony growth inhibition, which is too high a concentration for a systemic fungicide, which is normally used at 50 µg/ml or so.Barnwal (2003) reported that hexaconazole (0.1%) recorded the highest colony growth inhibition of S. vesicarium of 95.14 per cent among the six fungicides viz., thiram, hexaconazole, copper oxychloride, benomyl, mancozeb and thiophanate methyl.Hosen (2011) reported that iprodione was found to be the most effective fungicide to inhibit the radial mycelial growth of Botrytis cinerea and S. botryosum as compared to carbendazim and mancozeb 64 + metalaxyl 8 WG.In a growth chamber experiment to evaluate the efficacy of various fungicides against S. vesicarium, tebuconazole 25 EC (Folicur) @ 0.25 ml a.i.l -1 and procymidone 50 WP (Salithiex) @ 0.5 g a.i.l -1 reduced the disease severity by 90 per cent while fosetyl-Al 80 WP (Alliete) @ 2.0 g a.i.l -1 reduced the disease by 75 per cent in garlic (Basallote-Ureba et al., 1998).
The data presented in Table 5 revealed significant variation in colony growth inhibition among the different nonsystemic fungicides at different test concentrations.At 0.5 µg/ml, copper hydroxide 77 WP gave non-significant effect when compared to un-amended controls in terms of per cent colony growth inhibition.Mancozeb 75 WP (Indofil M-45) was found significantly superior to copper hydroxide 77 WP (Kocide) at all the test concentrations and recorded higher mean colony growth inhibition of 45.70 per cent as compared to copper hydroxide (32.01%).At 1000 µg/ml, mancozeb recorded only 74.44 per cent colony growth inhibition and thus was found less effective when compared to systemic triazole and strobilurin fungicides, as it did not record complete colony growth inhibition at such a higher concentration.Singh and Milne (1974) reported that mancozeb exhibited high fungicidal activity at or below 100 µg/ml a.i.against A. alternata and S. vesicarium, while all the other fungicides viz., bavistin, benomyl, captafol, captan, carboxin, chloroneb, chlorothalonil, dicloran, fuberidazole, oxycarboxin, thiabendazole, thiophanate methyl, thiram and zineb were not effective.Mishra and Gupta (2012) also reported that mancozeb @ 0.2 per cent was most effective in inhibiting the mycelial growth of S. vesicarium.

In vivo evaluation of different fungicides against onion purple blotch complex (PBC) in bulb and seed crop
The data presented in Table 7 and Table 8 revealed that all the fungicidal treatments were significantly superior to the untreated check in reducing the disease severity and increasing the bulb and seed yield.The analysis of variance showed significant variation in the disease severity and yield parameters among the different fungicidal treatments in both bulb and seed crops.Systemic triazole and strobilurin fungicides were found more effective as compared to non-systemic fungicides.
Tebuconazole 25 EC (Folicur) @ 0.1 per cent was found to be the most effective in reducing the disease severity (21.53 and 28.37%) which was followed by trifloxystrobin 25% + tebuconazole 50% WG (Nativo) @ 0. Highest fresh bulb and seed yield were recorded in trifloxystrobin 25% + tebuconazole 50% WG (18.54 t/ha and 3.34 q/ha) followed by tebuconazole 25 EC (18.12 t/ha and 3.12 q/ha) and difenoconazole 25 EC (17.64 t/ha and 2.85 q/ha), respectively.Similarly, highest fresh bulb size was recorded in trifloxystrobin 25% + tebuconazole 50% WG (5.56 cm) followed by tebuconazole 25 EC (5.51 cm) and difenoconazole 25 EC (5.43 cm), respectively.Trifloxystrobin 25% + tebuconazole 50% WG and tebuconazole 25 EC were found statistically at par with respect to fresh bulb as well as seed yield and size of fresh bulbs.All the fungicidal treatments resulted in significant increase in benefit: cost (B:C) ratio.The highest B:C ratio was recorded in tebuconazole (8.75:1 and 88.67:1) while the lowest B:C ratio was recorded in iprobenfos (0.46:1 and 3.41:1) in bulb and seed crop, respectively.The present findings revealed that despite of significantly higher disease control recorded by tebuconazole in bulb and seed crop, trifloxystrobin 25% + tebuconazole 50% (Nativo) was found to record numerically higher fresh bulb yield as well as size and seed yield.It could be attributed to growth promoting effects of azoxystrobin (a strobilurin compound) present in the commercial formulation.The present findings are in close agreement with those earlier reported by Upamanyu (1999), Deshmukh et al. (2007), Beig et al. (2008), Aujila et al. (2010), Aujila et al. (2013) and Bhatia and Chahal (2014) who have also reported the superiority of tebuconazole, propiconazole, difenoconazole and hexaconazole over conventional fungicides in controlling the disease.Similarly, the present findings are also in agreement with earlier reported by Vincelli (2002), Nelson and Meinhardt (2011), Henry et al. (2011) and Hill et al. (2013) who have reported the growth promoting effects of strobilurins in several plant species.

Evaluation of Method of Seed Production against Purple Blotch Complex in Onion
The data presented in Table 9 revealed significant variation among cultivars and different methods of seed production in terms of per cent disease index (PDI) and seed yield.However, no difference in disease incidence was recorded among the different cultivars and methods of seed production of onion.Significantly higher mean PDI (67.19%) was recorded in bulb-to-seed method as compared to mean PDI of 57.56 per cent in seed-to-seed method.
Similarly, significantly higher mean PDI (67.90%) was recorded in cultivar Punjab Naroya as compared to mean PDI of 56.85 per cent in cultivar PRO-6.Significantly higher mean seed yield (1.62 q/ha) was recorded in seed-to-seed method as compared to 0.73 q/ha in bulb-to-seed method.Similarly, significantly higher mean seed yield (1.34 q/ha) was recorded by cultivar PRO-6 as compared to 1.02 q/ha recorded on cultivar Punjab Naroya.It is evident from the data presented in Table 9 that the seed-to-seed method using cultivar PRO-6 recorded lower disease severity and higher seed yield as compared to other treatments.As the literature is silent regarding the effect of methods of seed production on disease incidence and severity, the present findings remain uncompared and new.

Fig. 2 :
Fig. 2: Percent inhibition in colony growth of Stemphylium vesicarium with different fungicides at their respective concentrations as follows: Arzanlou et al (2012))of S. vesicarium revealed that the mycelium of the fungus was initially sub hyaline, which later turned pale brown to dark brown.The hypha were septate, smooth irregularly branched measuring 4.02-6.13μm in thickness.Conidophores were scattered, unbranched, straight to flexuous, smooth, septate and pale to mid brown measuring 42.20-49.07μmx4.32-6.46μm.Conidiogenous cells were monoblastic, terminal, swollen and dark brown.Mature conidia were oblong to broadly ellipsoid, densely verrucose, golden brown to olive brown rounded at the ends and were 24.18-38.43μmx12.61-21.18μminsizewithlength to width ratios ranging from 1.64-2.43with1-3transverseand1-4longitudinalorobliquesepta and often constricted at one or more of the septa.These findings are in close conformity with those ofSimmons (1969),Ellis (1971),Basallote et al (1999)andArzanlou et al (2012)who reported that mature conidia are produced singly on the swollen apex of the conidiophores and are light to dark brown, densely verrucose, oval to ellipsoidal with transverse and longitudinal septa, and measured 25-42 μm x 12-22 μm.
The hypha were septate and irregularly branched measuring 4.52-7.26μm in thickness.The conidiophores arose singly or in groups, straight or flexuous pale to mid brown, septate and blunt at the tips.Basal cells of the conidiophore were somewhat wider than the tip cells,

Table 1 :
In vitro evaluation of different systemic fungicides against Alternaria porri

Table 2 :
In vitro evaluation of different non-systemic fungicides against Alternaria porri Fig. 1: Percent inhibition in colony growth of Alternaria porri with different fungicides at their respective concentrations

Table 3 :
ED50 and ED90 values of different fungicides against Alternaria porri

Table 4 :
In vitro evaluation of different systemic fungicides against Stemphylium vesicarium

Table 5 :
In vitro evaluation of different non-systemic fungicides against Stemphylium vesicarium

Table 6 :
ED50 and ED90 values of different fungicides against

Table 7 :
In vivo evaluation of different fungicides against onion purple blotch complex in bulb crop during Rabi 2014-15 *Per cent disease index ** Per cent disease control Figures in parentheses represent per cent disease index in arc sine transformed value Values in the column with different superscripts are significantly different (p≤0.05) according to Tukey's HSD test

Table 8 :
In vivo evaluation of different fungicides against onion purple blotch complex in seed crop during Rabi 2015-16 Percent disease index ** Percent disease control Figures in parentheses represent percent disease index in arc sine transformed value Values in the column with different superscripts are significantly different (p≤0.05) according to Tukey's HSD test. *

Table 10 :
Evaluation of method of seed production against purple blotch complex in onion during Rabi 2015-16