Neem Leaf BioMass And Leaf Extracts

Neem leaves have been known for centuries to contain compounds that are repellent to insects, thus serving to protect neem foliage from insect feeding, but they also contain anti-fungal compounds which can be evidenced by either direct incorporation of the leaves into soil as organic amendments (green manure) or by using extracts of them. The concentration of bioactive compounds in the leaves is probably lower than in the fruits, but still of interest for agricultural purposes.

Neem Leaf Biomass

It has been demonstrated that the direct incorporation of either fresh or dried leaves as a green manure can result in the population decline of several soilborne pathogens. A significant reduction in will incidence, caused by Phytophthora Capsici, was achieved in belelvine (Piper betle) when dried neem leaves were applied near the base of the vine. Population of Pythium Aphanidermatum were reduced following incorporation of green neem leaves (5% w/w) into infested soil (SINGH and PANDEY, 1966). Similarly, the application of chopped neem leaf amendment reduced the total fungal population in tomato rhizosphere soil. However, neem as a green manure prolonged the vialbility of sclerotia of Curriculum Sasazaki over controls while increasing the total microbial population in the trested Soil.

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Seed Cake

In greenhouse experiments NAIDU et al. (1980) treated rice clip-inoculated with a virullent strain of Xanthomonas Oryzae with uncoated urea, coal-tar-coated urea, and nem cake with coal-tar-coated urea and comapred the results of the untreated control. There was no reduction in leison formation. Compared with the control, all various had higher values for leison length, the symptom parameter for X. oryzae, and higher values were obtained for higher additional doses of nitrogen and split dosages with a base dose. The highest rates were for neem cake with coal-tar-coated urea and an additional split dosage of 200kg N/ha which also led to the tallest plants to the experiment. Undoubtedly the total amount of fertilizer played an important role, as did the influence of neem cake on nitirification - its inhibition or recardation in the soil. The influence was studied in many experiments. MISRA and CHHOKAR explained the inhibition of denitrification by the inhibition of Nitrosomonas and Nitrobacter spp.

Another strategy to save nitrogen is by the use of seeds treated with Rhizobium spp Neem cake did not affect module formation, except at high doses, which had some effect on Rhizobium spp but not on grain on master yield. (The authors did not say, what the does was but it has been more than 7 ppm on the soil weight basis).

In experiments to evaluate the inhibitory effect an Fusarium Udum of soil incubated with neem cake in vitro B. Thuringiensis was frequently detected in the lylic zones of fungus growth. The microbial activity in decomposing neem cake, especially of bacteria, was described by PETHAMBARAM the increase in the population of bacteria in neem cake amended soils was mentioned by JEYARAJAN et al.

ESWARAMURTHY et al.(1993) reported reduction of symptoms in rice caused by X. Compestria pv. oryzae and a considerable increased in yield when neem cake extract was sprayed twice at a one-weel interval. Repetitions of the treatment also increased yields compared with control.

In laboratory experiments CHANDRAMOHAN and MOSES (1996) demonstrated that extracts of neem cake were effective against X. Compestria pv. cltl with extracts being better than awueous extracts.

Seed Oil

As early as 1917, the antibiotic in vivo effectiveness of margosates obtained from neem oil was proved by CHATTERJI and RAY in combating syphilis and other skin diseases. CHOPRA et al tested oil from neem seeds against a number of bacteria causing diseases in humans and animals. The oils were added directly to the substrates in vitro at different dilutions, Neem oil was very efficient against a human strain of the same bacterium showed some growth at the dilution of 22,000. P.pestis and S. aurex showed some growth already at a dilution of 5,000, while S.schotemelleri and K. pneumoniac showed normal growth at this concentration.

PATEL and TRIVEDI tested neem oil in a cup-plate method measuring the inhibition zones. Unfortunately, they did not use an antibiotic standard and compared only the inhibition of different oils with that of the emulsifier used, which have no inhibition. Neem oil showed the weakest inhibition when compared with oils of karanja, malkanguni and darudi. No inhibitions by neem oil was found against Pseudomonas Pyocyanea and Proteus Vulgaris. When compared with darudi oil, which in most cases ws the best inhibitor, neem oil gave only 63% inhibition for two strains of S. aureus genes var. aureus and M.pyogemer, B. subtilis, S. paratyphi and E. coli. Against another strain of S. aureus neem oil gave 65% inhibition; for C. diphtheriac, 67%; for S. schotmuelleri 74% and for S. typhosa, 78%. The maximum inhibition zone for darud and neem oil was 32 and 20 mm, respectively, achieved with S. aureus, the minimum was 21 and 14mm, respectively with C, dlphtheriae. However, in serial dilution tests darwal oil was less effective then neem oil in reaching the inhibiting concentration for S. aureus with 0.4% and 0.3% and for S. typhosa with 0.8% and 0.4%.

The antibacterial activity of neem oil in vitro was tested also by RAO et al. using many strains of Pseudomonas Aeruginosa(55), S.aureus, E. coli, Proteus spp, and Klebsiella Pneumoniae (?)(5). Inhibition with pure neem oil detected to the extent of 85%, 94%, 94%, 100% and 80% of the strains respectively. The effect was good for 11%, 12%, 21%, 8% and 60%, medium for 42%, 59%, 45%, 46% and 20% of the strains, and a week reaction was observed in 33%, 22%, 21%, 42% and 0% respectively. However inhibition was lost quickly, when the oil was diluted. In I/64 dilution none of the Pseudomonas Aeruginosa strains only 20% of S. aureus, E. coli ans Proteuis spp and 25% of K. aerogenes strains were still sensitive. SINGH et al. described in 1974 an antibiotical effort of neem oil on E. Coli.

Neem oil extracted with petrol either in a Soxhlet appratus showed in vitro inhibition of Bacillus pumilus, B.substiz, S. typhasa, S. paratyphi and S.aureus using filter disc test, but it is difficult to quantity of activity as a results of controls were given.

Commercial Products

Little inforamtion is available on the effect of commercial neem products on viruses. AH-MED(1988) reports that 'Wellgro' a product of ILTD is used by farmers to control TMV in the West Godavari district. The effectiveness of the treatement seemed to be as good as spraying neem leaf extracts, but no detailed data or quantities were given. Other commercial products are used in the control of vector as vector repellents, like 'RD-Repelin' which is a mixture of extracts of neem(A.Indica), karamja(Pongamia Pinnata), castor (Ricinus Communis), Mahua (Madhuca Indica) and sesame(Sesamum Indicum). In addition to a repellent effect against the pea aphid. Acyrthosiphon Pisum, which did not prevent virus transmission, a delayed symptom expression for zucchini yellow mosaic virus(ZYMV) was observed in 81% of the test plants. The mixture of ingrediants does not, however allows us to contribute this finding to a particular plant extract. No influence on virus replication by 'RD-Repelin' was found, because the ELISA-readings of untreated and treated plants did not differ significantly.

ADDING NEEM TO SOIL

The use of neem cake as an amendment to agricultural soils is a well known practice in India, mainly to exploit the fertilizer properites of this material but also in combination with synthetic chemicals for plant protection. The influence on viruses has not been studied yet in details. Some experiments with neem cake(NC), neem seed kernels(NSK) and dried neem leaves(NL), in the field and under greenhouse conditions yielded very different results, depending on the virus and host plant used. In field experiments, when neem was added to 1% of the upper 10cm layer none of the neem products used was able to prevent infection of zucchini with watermelon mosaic virus. In a second experiment under much higher infection pressure, the results in the neem plots were much better than in the control, and a delay in symptoms expression was significant for NSK powder and to some extent for NL powder. In a greenhouse experiment with artificial inoculation, in which the zucchi plants were planted in prickout pans with soil containing 1% neem, the inhibition was low but marked: only 80% of the zucchini plants treated with NS, 85% with NC and 82% with NL, but 100% of the control plants became infected, as determined by symptom expression.

When hups were used as test plants with the same method, a reduction of the ELISA reading for hop mosaic and hop latent catlaviruses was obtained in the plots treated with crushed NSK and NL, but the differences from the control were not significant. Another system used for testing this type of application was Prunus Necrotic ringspot virusand cucumber planted in 10cm pots of five plants each. In this artificial system the use of neem did not prevent virus infection after artificial inoculation, but there was a reduction in the number of plants successfully inoculated and with increasing amounts of neem added(0.5%, 1%, 2,5%) there was a significant reduction in symptom expression. This corelated with the average vales for the ELISA-readings. The efficacy increased from Soxlet extracted NC to NC to NSK, but 5% NSK induced also phytotoxic reactions, measurable as reduced growth and fresh weight.

Pure Ingrediants

VERMA(1974) demonstrated inhibition of PVX in vivro and in vivi using C, Amaranticolor as a single lesion host. When allowed to preincubatable for 15-20 min, 44% and 63% inhibition of lesion formation was obtained using nimbin and nimbidin, respectively. With pretreatment of the test plan by spraying nimbin and nimbidin even better results were obtained, when the treatment was carried out 12 h or more before in oculation. The inhibitory effect of the treatments p.i. also increased with more time after the infection process and reached 43%(nimbin) and 35%(nimbidin) when carried out 24 h p.i. Unfortunately, VERMA did not mention which of the three concentrations used in the first test used to perform the pre- and post- inculation tests. A pre inculation root treatment for 24 h did not lead a reasonable inhibition when tomatoes were used, but with C. amaranticolor, local lesion formation was reduces using a mixture of nimbidin and nimbin at 250 ppm. Higher concentrations mostly  resulted in phytotaxics reactions of both test plant species. Experiments by the author using aza resulted in high or low inhibition, but it was never significally different from with the methonal control, indicating the aza has no significant antiviral properties that the methonolic solution seems to be an inducer of resistance. Similar results were obtained with methonolic extracts of neem representing an earlier stage by the methonal control, but in some experiments higher concentrations of extracts tended to have stronger effects.

SEED OIL

Seed Oil

In field experiments conducted in Tamil Nadu (India) 1% neem oil sprays reduced significantly ring mosaic disease of peanut caused by ToSWV. The effect of oil application seemed to be twofold; the incidence of the viral vector Frankliniella Schultzei was reduced and an antiviral activity was diagonosed under laboratory conditions. Unfortunaetly the numbers did not give the details of these laboratory experiments that "antiviral activity was measured by symptoms, When Vigna Sinensis was used as the indicator plant, neem oil completly inhibited lesion formation. A reduction in urd bean leaf crinkle infection in Vigna Mungo was demonstated by SABITHA and JEYARAJAN with sprays of neem oil(1% 5%) but it did not exceed 40% and it cannot be excluded that this effect was only due to an inhibition of the virus transmission i.e the vectors. As no further details were given, it remains unclear which of the crinkle viruses of the urd bean found in India is meant the ULCV transmitted by Henosepilachna Dodecastigma the applied transmissible virus or the whistlefly-transmissited virus. All three vector groups can be affected by neem, as discussed later. In experiments with ilarviruses and cucumbers, topical application of neem oil had no effect on the number of infected plants or severity of symptoms ROYCHOUDARY and JAIN gave a diffferent interpretation for their results with mechanical inoculations of different host species with TMV, saying that virus multiplication is inhibited by neem oil.

Seed Kernel Extracts

AQUEOUS EXTRACTS

Aqueous extracts of neem seed kernels, when preincubated with TMV in laboratory and greehouse tests, gave varying results. Strong inhibition in one test was followed by increased numbers of lesions in the next. In general concentrated virus inocula led to less inhibition than more diluted inocula. Using systemise hosts like N.labocum cv.Java, a delay in symptom development was observed, along with much better growth of the neem-treated plants. Using apple mosaic ilavirus and cucumber as a model system, topical applications of aqueous extracts pre and post infection did not lead to a reduction of the symptoms.


METHONOLIC EXTRACTS

Methonolic extracts, as discussed below for Azadirachtin(a2a), bear the problem of methonal itself, either as a denaturating component or as a resistance inducer. Preinoculation applications of methonolic extracts inhibition when the results were compared with the methonal control, but the data differed widely among the various concentrations of neem and there was no correlations with the inhibition values. When the results of the pretreated halves of the leaves were comapred with the untreated ones, there were always fewer Jesions but the difference was not always significant and again there was no correlation with the neem concentration. When no methanol control was used, positive results were always reached, with the above mentioned limitations. When the extract were used only for co-inoculation, a significant reduction - with values above 90% - was obtained again when compared with pure virus control coly. Also the leaf halves inoculated with the virus alone showed nearby the same reduction as the other half with relatede virus. However, in this experiment, no menthol control was used.

BARK AND ROOT EXTRACTS

MURTY described a 87% reduction of TMV infection of N. gluntinoza using an aqueous extract of the bark of the neem tree. In experiments by the author, a methonolic extract from neem bark from Senegal resuspended with the microvolume of methonol and diluted with buffer, gave similar results when preincubated with the virus. Using the half leaf method, the number of lesions also decreased on the control-half of the leaves inoculated without extract, indicating a systemic effect when cv. Xanthi NN used. This effect was less significant using cv. White Burley as test plant. The bark extracts from different sources behaved very similarly. In an additional test, the bark from two different sources in Senegal was compared. With 250 ppm of extract, 57% and 62% inhibition respectively, was obeserved. Inner bank and wood was less effective, with only 43% inhibition. Compared with a methyl tert, buthylether extract of dried leaves from Senegal which gave 74% inhibition, all other bank and wood preparation were weaker. This activity was even weaker when Datura Stramonium was used as the test plant instead of tobacco; the inhibition reached , only 34% and 27% for the outer bank, 36% for the inner bark and wood, and 33% for the left extract. Symptom inhibition was significant only for one of the bark-samples when Nicotiana Benthamiana was used as systematic test plant. When the preincubation period was extended to 5 d, the inner bark and wood extract gave a weak inhibition of <10% and the outer bark, and the leaf extracts showed even an increase in lesions formation on the half-leaf inoculated withn the pretreated inoculum.

In general, there was a great variability in the values obtained for individual leaves when pretreating virus with extracts. Even in control experiments using the half-leaf method, differences of up to 49% could be observed when both parts were inoculated with the same pretreated inoculum, an effect never observed with the virus alone. This indicates that experiments with a low number of replications will occasionally lead to erratic results. Inhibition - with lower than with whole plants - was obtained using leaf discs and pretreated inoculum. When an inhibitor-seaked filter paper was placed on leaf discs. Again the effect could not be enchanced by using more inhibitor-discs. These experiment were performed with N. tabacum. When Nicotiana rustica was used, no inhibition was observed and with D.Stramonium only 30% inhibition was achieved under the same conditions.