Evaluation of Botanicals for Managing Leaf Rust of Castor Caused by Melampsora ricini

Description of the Study Area

The experiment was conducted at Melkassa Agricultural Research Center (MARC) from 2006-2010 E.C. MARC is found 117 kms away from Addis Ababa and 17 km to South-East of Adama in the East-Shewa Zone of Oromia Region. It is located 8°24'N latitude and 39°12'E longitude at elevation of 1500 meters above sea level and a mean annual rainfall of 770 mm.

Planting of Castor Seeds in the Green House

A mixture of sterilized sandy clay loam soil, decomposed animal dung and sand (3:2:1 ratio) was autoclaved at 121°C for 2 h and filled into plastic pots (20 cm ×15 cm). Susceptible variety of castor (Hiruy) was used for the study. Two Seeds of castor per pot was planted on sterile soil in plastic trays containing 1200 cc of sterilized soil with 1:2:3 proportions of sand, compost and clay, respectively in greenhouse and kept on benches. The plants were allowed to grow for 6 weeks in a greenhouse while maintaining the soil field capacity at 26 ± 2°C and 50 to 60 % relative humidity.

Preparation of Plant Extracts

leaves, seeds, flower and bulb of 8 plants, namely Neem (Azadirachataindica), Garlic (Allium sativum), Pyrethrum (Pyrethrum Parthenium),Lantana weed (Lantana camara L.), Australian Fever Tree (Eucalyptus globules), Stinking Roger (Tagetes minuta), Congress weed (Parthenium hysterophorus L) and Jatropha (Jatropha curcus) (Table1) were collected from different parts of the country and test for their efficacy in reducing the mycelia growth of in vitro using , the poisoned food technique.

They were sun dried for 3 days then crude extract of each plant was prepared as a solvent. The extraction technique is used as a modification of Ruch and Worf method. One hundred and fifty gram of the dried material of each plant was soaked in 500 ml of water with constant stirring for 30 min and then maintained at room temperature for 24 h before being filtered. The soaked plant material was filtered with the help of a very fine and clean piece of cheese cloth separately for each plant. The filtrates were preserved in glass bottles in a refrigerator at 4°C for further use. The plant extract will be further diluted to have 20 % concentration.

Isolation and Preparation of Spore Suspension of a Leaf Rust Disease (Melampsora ricini)

The diseased leaves having distinct symptoms with Melampsora ricini was collected from the castor farm at MARC, in sterilized polythene bags and brought to Crop Protection Laboratory, MARC for culturing, isolation and identification. The diseased leaf having distinct leaf spot symptoms was collected and the identification of causal pathogen was observed with microscope.

For preparation of inoculum (spore suspension), sterile distilled water was added and dislodging spores gently with a sterile glass rod. The suspension was subsequently filtered through 4 layers of cheese cloth in order to remove the mycelia and the concentration of spores is determined by means of haemocytometer. The concentration of spores was adjusted to 1x 105 spores per ml with sterile distilled water prior to the inoculation of castor leaves.

**Experimental Design and Treatments Application**

A total of 10 treatments were used, namely; crude extracts of Neem (Azadirachta indica), Garlic (Allium sativum), Pyrethrum (Pyrethrum Parthenium), Lantana weed (Lantana camara L.), Australian Fever Tree (Eucalyptus globules), Stinking roger (Tagetes minuta), Congress weed (Parthenium hysterophorus L), and Jatropha (Jatropha curcus) were evaluated in greenhouse conditions. There were also two controls. Treated control/ standard check (mancozeb) and untreated control (neither rust nor the botanicals was applied. The spore suspension of the pathogen containing 1x 105 spores/ml was sprayed on one and half months of castor seedlings. Then, the botanicals each at 20 % concentration was sprayed on infested plants after 72 h of pathogen inoculation and continued for two months at 15 day interval. The treatments were arranged in randomized complete block design (RCBD) with three replication.

**Data to be Collected**

Percent disease control (PDC) is calculated using the formula given by Wellker (1988). Estimation of disease severity is made by visual disease assessment key. Disease intensity was assessed using 0 – 4 points rating scale, where 0 = no disease (healthy); 1 = 1 to 25%, 2 = 26 to 50%; 3 = 51 to 75%; 4 = 76 to 100% leaf areas infected, using the following formula:

$$\text{PDC} = \frac{\text{DC-DT}}{\text{DC}}$$

**Field evaluation of effective botanicals against leaf rust disease (Melampsora ricini) of castor plants**

Field Preparation: Experiment was conducted in a field with a history of leaf rust disease (Melampsora ricini) at MARC; susceptible variety of castor (Hiruy) was planted in row to row and plant to plant distance of 75cm and 50cm, respectively in the field of sericulture for this experiment. The fertilizer, DAP and Urea were applied as recommended whilst, watering and hand weeding was done as recommended.

**Experimental design and Treatments application**

Only those effective plant extracts i.e., Azadirachta indica (Neem), Allium sativum (garlic), Pyrethrum Parthenium (phyrethrum), Lantana camara L. (Lantana Weed) during greenhouse experiment were further investigated under field condition. Untreated control and the standard chemical were applied in the field as in the green house. The experiment is laid down in a randomized complete block design (RCBD), with three replications. The treatments will be applied at an interval of 15 days starting from the first appearance of the disease. A guard row was kept in between the treatments to prevent the drift while spraying.

**Data to be Recorded**

Percentage of disease incidence and severity were taken and calculated based on the following formulae suggested by Mc Kinney [7]:

$$\text{Incidence}(I) = \frac{\text{Number of affected plant units}}{\text{Total number of plant units (healthy and affected units assessed)}} x 100$$

$$\text{Severity}(S) = \frac{\text{Area of plant tissues affected by pest}}{\text{Total area of plant (tissue)}} x 100$$

Other agronomic data such as: plant height, production of leaves, fresh leaf weight and dry leaf weight were assessed.

Efficacy of Plant Extracts Under Greenhouse Condition

The effect of plant extracts against leaf rust infestation in castor was studied in green house. The extracts were applied as spray method during inoculation. The results of the experiment showed that all the eight botanical extracts revealed significant differences (P<0.05) as compared to the control (Table 1).

Among botanicals, minimum disease incidence and severity on leaves (18.7% and 24%) were observed in neem, which was at par with garlic (20% and 26%), Pyrethrum (23 % and 30%) and Lantana weed (21% and 27%), respectively. However, similar results of incidence were recorded from Australian fever tree (30%), stinking rogus (32 %), congress weed (33 %) and jatropha (31 %). The extracts able to reduce the severity of the disease ranged from 10 to 52 % (Table 1). The standard fungicide (Mancozeb) showed highest reduction in disease incidence (63%) and severity (64%). On this study applying a foliar spray showed high efficacy in reducing the leaf rust infection and neem extract was the most effective treatment as compared to botanical extracts and it reduced disease incidence and severity by 46.6 % and 52 % respectively (Table 2). The increasing serious problems of resistance and residue to pesticides and contamination of the biosphere associated with large- scale use of broad spectrum synthetic pesticides have led to the need for effective biodegradable pesticides with greater selectivity. This awareness has created a worldwide interest in the development of alternative strategies, including the discovery of newer pesticide [9].

There were significant differences (P < 0.05) among treatments in affecting, fresh and dry leaf weight as compared to treated control. Maximum fresh leaf weight (48 gm) and dry leaf weight (9.6gm) per plant were recorded from mancozeb. Among botanicals neem recorded highest fresh (40 gm) and dry leaf weight (8 gm) per plant. John and Hebs also reported that bare root dip of brinjal seedlings in neem leaf extract significantly reduced the gall index and increase the plant growth parameters significantly [10, 11]. Soil application of neem and caltropis leaf extract reduced the root knot nematode infestation in cowpea.

Neem, Azadirachta indica, is one of the most versatile trees with regard to germicidal activity against an array of microorganisms. Its leaves, bark, and seed kernels can be used in nematode management. Various compounds such as nimbin, nimbidin, azadirachtin, salannin, thionemon, and meliantriol occur in the seeds, leaves, and bark of neem in high concentrations and are responsible for the tree’s antimicrobial and nematicidal activity. Powder from the seed kernels and leaves has been found to be suppressive against some diseases [11].

Sindhan and Hooda, reported bulb extracts of Allium sativum (garlic) and A. cepa (onion) more effective than eucalyptus leaf extract for the control of white rust of castor [12]. However, all plant extracts improved fresh and dry weight of leaves as compared as treated control. It is evident from several reports that plant extracts are effective bio control agents against a wide range of plant pathogens [13].

Promising botanicals verified during greenhouse evaluation i.e neem, garlic, pyrethrum and lantana weed were selected and further evaluated under field condition.

For the field experiment, only those effective botanicals during greenhouse experiment were investigated. All the treatments were significantly superior to untreated control for rust severity on leaf (Table 3). All botanical extracts applied on rust infested plots significantly (P ≤ 0.05) decreased the severity of rust disease over untreated control plots (Table 3).

Among considered botanicals, minimum incidence (26.36 %) and disease index (29.45 %) were recorded from neem. Minimum disease index on leaves (29.45 %) was observed in neem, which was at par with garlic (33.33%), pyrethrum (40.56 %), lantana weed (47.22 %) and mancozeb. The result indicates spore germination was affected by some of the plant extracts but complete inhibition was not achieved by any of them. The neem plant is the most well-known example and its various parts, namely, leaves, crushed seeds, powdered fruits, oil, and so forth, have been used to protect stored grain from infestation [14]. The neem oil and kernel powder gave effective grain protection against stored grain insect pests like Sitophilus oryzae, Tribolium cataneum, Rhyzopertha dominica, and Callosobruchus chinensis at the rate of 1 to 2% kernel powder or oil [15]. Yadava and Bhatnagar reported that a dried leaves of Azadirachta indica have been mixed with stored grains for protection against insects [16]. Azadirachtin is an active principle from the neem plant, which is an effective grain protectant against insect infestation.

The present study has demonstrated the antifungal potential of plant extract that corresponded with the There were no significant differences (P<0.05) in plant height, number of primary branches, stem thickness, number of leaves, leaf area as well as internode length. However, significant differences in fresh and dry leaf weight and number of capsule were observed between treatments. Highest fresh and dry leaf weight were recorded from mancozeb (330gm and 85 gm neem) a neem (320gm and 70 gm) respectively. Whilst, the least fresh and dry leaf weight were recorded from control (210 gm and 38 gm) respectively (Table 4). Javed et al reported that neem is one of the most effective agents against root knot nematode on the roots of tomato, neem formulations have been applied against root knot nematodes development of the second stage juveniles (J2) as soil drenching [20]. Fadzirayi, et al. stated that garlic disrupts nematode mobility, feeding and reproduction, the latter being a plausible explanation for the low reproduction rate [21, 22, 23].