Evaluation of calcium sulphate, potassium silicate and moringa dry leaf powder on Meloidogyne incognita infecting tomato plant with reference to N, P, K, total phenol and cholorophyl status under greenhouse condition

A pot randomized complete block design experiment was carried-out to evaluate the impact of calcium sulphate or potassium silicate or moringa dry-leaf powder either alone or combined as double or triple treatments along with oxamyl or the three items + oxamyl 24% L as tetra application in comparison with oxamyl at the recommended dose against Meloidogyne incognita infecting tomato plant cv. 9065 FI under greenhouse conditions (25±3C). All tested treatments improved plant growth criteria and reduced nematode parameters as well. Moringa dry leaf powder singly surpassed other tested single treatments in the increment values of total plant length, number of leaves and branches per plant; total plant fresh weight and shoot dry weight, and also accomplished the highest percentage reduction of nematode parameters with the maximum values of final nematode population, number of galls and egg mass/plant, respectively. Among the dual treatments, moringa dry leaf powder plus calcium sulphate at their half doses overwhelmed other double treatments in the percentage increase values of such plant growth characters of tomato and achieved the highest reduction values of final nematode population, number of galls and egg masses. Likewise, the same trend was evident as the two compounds of moringa dry leaf powder plus calcium sulphate mixed with oxamyl at 1∕3 each. Moreover, a similar trend was also strongly observed in the case of applying oxamyl to that tested three components as 1∕4 each which gave the high synergistic action by showing the highest recorded values of plant growth criteria and also recorded a high synergistic action in diminishing nematode criteria as well as the highest percentage increase value of total phenol (23.70%). The C/N ratio of the tetra treatment gave the least value of this item (16.03:1), while oxamyl had 19.67:1 vs. 22.88:1 for nematode alone, respectively.


INTRODUCTION
Root-knot nematodes (Meloidogyne spp.) are one of the most wide spread and damaging agricultural pests in the world causing an estimated US $100 billion loss/year worldwide (Oka et al., 2000).They are widely distributed in cultivated areas of Egypt causing remarkable crop losses.Tomato is preferable host to several species of root-knot nematodes.Biocontrol agents applied singly are not likely to perform consistently against all parasitic nematodes under soil environmental conditions.Different mechanisms of control may be dissimilarly influenced by environmental conditions; and it is possible that if multiple mechanisms are involved under a certain set of conditions, one mechanism may compensate for the other (Guetsky et al., 2002).So, the control achieved by biocontrol agents with several distinct mechanisms of control may be additive or synergistic.The approach of combining biocontrol agents to manage various soil borne pathogens including plant parasitic nematodes has been investigated extensively ( Hojat et al., 1998;Pierson and Weller, 1994;and Siddiqui and Mahmood, 1993).Integrated nematode management using several control techniques that is soil amendments and bioagents with minimal use of nematicides received recently great attention among nematologists, providing effective control measures against the target nematode by keeping nematode population densities at the safe level and avoiding environmental pollution (AL-Ghnam, 2011).The objective of the present work was to study the impact of calcium sulphate or potassium silicate or moringa dry-leaf powder in comparison with oxamyl on plant growth response of tomato plants infected with M. incognita under greenhouse conditions.

Source of nematodes
Second stage juveniles (J2) of M. incognita (Kofoid & White) Chitwood, were obtained from a pure culture of M. incognita that was initiated by a single eggmass propagated on coleus plants, Coleus blumei in the greenhouse of Nematology Research Unit (NERU) Agricultural Zoology Department, Faculty of Agriculture, Mansoura University, Egypt, where this work was carried-out.Second stage juveniles (J2) were extracted from soil of coleus plants by sieving and modified Baermann technique (Goodey, 1957) counted in a Hawksely counting slide under x 10 magnification then calculated for each 1 ml of the nematode suspension and recorded for preparing nematode inoculation of this work.
Each treatment was replicated four times.Plastic pots were arranged in a randomized complete block design and irrigated with tap water as needed.Plants were harvested 45 days after nematode inoculation, and plant growth criteria that is shoot and root lengths, number of leaves and branches per plant and fresh weights, as well as shoot dry weights were determined and recorded.Number of M. incognita (J 2 ) in 250 g of soil/pot were extracted by sieving and modified Baermann technique (Goodey, 1957) counted in a Hawksely counting slide under x 10 magnification then calculated for each pot and recorded.Infected roots of each plant per treatment were washed with tap water, fixed in 4% formalin for 24 h and stained in 0.01 lactic acid-fuchsin (Byrd et al., 1983) and then examined for the number of galls, developmental stages, females and egg-masses.The root gall index (RGI) and egg mass index (EI) were estimated according to the scale given by Taylor and Sasser (1978) as follows: 0= no galls or egg-masses, 1= 1-2 galls or egg-masses, 2= 3-10 galls or eggmasses, 3= 11-30 galls or egg-masses, 4= 31-100 galls or eggmasses and 5= more than 100 galls or egg-masses.The obtained data were subjected to analysis of variance (ANOVA) (Gomez and Gomez, 1984) followed by Duncan's multiple ranges to compare means (Duncan, 1955).

Chemical analysis
Samples of tomato dried leaves were ground, wet, digested and their nitrogen (N), phosphorus (P), potassium (K) contents were determined according to Kjeldahl methods (A.O.A.C,1980).

Chlorophyll content
Representing sample from the upper fourth leaf of tomato plant / *Corresponding author.E-mail: elsherifmohammed@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License treatment were obtained at 75 days after sowing and both chlorophyll a and b were determined in mg/g F.W following the equations used for the calculation of pigments content according to Goodwin (1965).

Determination of total phenols
Total phenols were determined after harvesting tomato fresh leaves bases using the Folin-Ciocalteu reagent method (Kaur and Kapoor, 2001).Total content of phenolic compounds in plant ethanolic extracts was calculated as catechol equivalents by the following equation: Where, T=Total content of phenolic compounds, in mg of catechool/100 g of fresh weight material.C = The concentration of catechol established from the calibration curve, in mg/ml and V= the volume of extract in ml; m =The weight of pure plant ethanolic extract in g.

RESULTS AND DISCUSSION
Tables 1 and 2 verify the impact of calcium sulphate or potassium silicate or moringa dry-leaf powder either alone or mixed as double or triple treatments along with oxamyl 24% L or the three elements + oxamyl as tetra application in comparison with oxamyl at the recommended dose against M. incognita infecting tomato plant cv.9065 FI under greenhouse conditions (25±3°C).Obviously, results indicated that all tested treatments improved plant growth criteria and reduced nematode parameters as well.
Among single applications, plant that received moringa dry leaf powder surpassed other tested single treatments in the increment values for total plant length (57.8%), number of leaves (13.3)% and branches per plant (28.6)%; total plant fresh weight (41.12%) and shoot dry weight (40%) followed by calcium sulphate in this respect compared to nematode alone (Table 1).
Moreover, among the dual treatments tested in this study, moringa dry leaf powder plus calcium sulphate at their half doses overwhelmed other double treatments in the percentage increase values of such plant growth characters of tomato plant that is total plant length (94.25%), number of leaves (55.5%) and branches (60.71%), total plant fresh weight (70.71%) and shoot dry weight (80.0%), followed by that of ½ (calcium sulphate + potassium silicate) and then ½ moringa dry leaf powder + potassium silicate), respectively.Likewise, the same trend was evident as the two compounds of moringa dry leaf powder plus calcium sulphate mixed with oxamyl at 1∕3 each since this integrated treatment achieved the highest percentage increase values of total plant length (124.26%),number of leaves (61.6%), and branches (67.85%), total plant fresh weight (110.13%) and shoot dry weight (105.2%)followed by that of ⅓ (potassium silicate + moringa dry leaf powder + oxamyl ) and then 1∕3(potassium silicate + calcium sulphate +oxamyl)in such plant growth parameters, respectively.It is interesting to note that a synergistic effect to the increments values of tomato plant growth positively occurred for the three tested items added at ⅓ each with values of 111.46, 61.6, 71.42, 113.26 and 114.8% for plant length, number of leaves and branches∕plant, total plant fresh weight and shoot dry weight, respectively compared with nematode alone.Moreover, similar trend was also strongly observed in the case of applying oxamyl to that tested three components at ¼ each as 1∕4 (moringa dry leaf powder + calcium sulphate + potassium silicate + oxamyl) treatment which gave the high synergistic action by showing the highest recorded values of such tomato plant growth criteria in this study, since its values were 146.84, 66.6, 114.28, 127.04 and 122.8% for plant length, number of leaves and branches/plant ,total plant fresh weight and shoot dry weight, respectively (Table 1).It is worthy to note that oxamyl as a systemic nematicide gave considerable values of percentage increase values of tomato plant growth characters, since its values amounted to 93.6, 34.6, 28.57, 78.53 and 81.2% for plant length, number of leaves and branches / plant, total plant fresh weight and shoot dry weight, respectively, comparing to nematode alone (Table 1).Moreover, plant free of nematode and receiving none of the tested components gave reasonable values of such tested tomato growth characters, since its values were 12.89, 4.4, 7.14, 4.32 and 8.0% for plant length, number of leaves and branches ∕ plant, total plant fresh weight and shoot dry weight, respectively (Table 1) .
Data presented in Table 2 reveal the influence of calcium sulphate, potassium silicate and moringa dry leaf powder either alone or mixed as dual or triple treatments along with oxamyl or three items plus oxamyl as tetra application compared to oxamyl at the recommended dose on reproduction and development of M. incognita infecting tomato plant cv.9065 F1 under greenhouse conditions.In general, results indicate that all tested treatments obviously diminished M. incognita tested criteria that is number of juveniles in soil, developmental stages, root galling, females and egg masses on root system as compared to those of the inoculated untreated cheek.It is interesting to observe that the tested double or triple or tetra application treatments gave better results than single ones did.In the meantime among the single applications, moringa dry leaf powder accomplished the highest percentage  when the dual applications applied separately along with oxamyl as triple treatment, an obvious synergistic action of such triple application was clear in the resulting more reduction percentage of nematode criteria.For instance, plant receiving treatment containing ⅓ (moringa dry leaf powder + calcium sulphate + oxamyl) surpassed over other tested triple treatments in reducing final nematode population (90.5%), number of galls (88.90%) and egg masses (93.33%), followed by that of ⅓ (potassium silicate + calcium sulphate + oxamyl), respectively, compared to nematode alone.
However, plant that received the three compounds as triple treatment without oxamyl [⅓ (calcium sulphate + potassium silicate + moinga dry leaf powder)], gave a considerable reduction percentage of final nematode population (87.3%), number of galls (91.66%), and egg masses (92.59%), respectively.Moreover , when oxamyl was added to this triple treatment that contained four components as tetra application [¼ (calcium sulphate + potassium silicate + moringa dry leaf + oxamyl)], a high synergistic action was obviously recorded in diminishing final nematode population, number of galls and egg masses with values of 91.5, 92.53 and 94.81%, respectively compared to nematode alone.It is worthy to note that oxamyl as a systemic nematicide gave the highest, percentage reduction of final nematode population (93.51%), number of galls (97.04%) and egg masses (98.51%), respectively compared to nematode alone and ranked first in this respect.Likewise, signification results were observed between egg masses indices of all tested treatments and nematode alone, since they ranged from (4) for single applications to (3) for the double ones to (2) for the triple treatment to (1) for oxamyl vs 5 for nematode alone.Similar trend was evident in the case of gall indices of tested treatments since they ranged from 4 for single and some of dual ones to 3 for triple treatments and 2 for four components as tetra treatment with oxamyl vs. 5 for nematode alone (Table 2).
Also, nematode reproduction factors under the stress of potassium silicate, calcium sulphate and moringa dry leaf powder solely or mixed as binary or triple alone or along with oxamyl or as tetra treatments in comparisons with oxamyl at the recommended dose on tomato plant were adversely affected.Such rates ranged from 0.94 to 0.21 vs. 2.43 for nematode alone.Namely, the treatment containing ¼ (potassium silicate +potassium sulphate +moringa dry leaf + oxamyl) had the lowest rate of reproduction 0.21 whilst that of potassium silicate alone showed clearly the highest (0.94), respectively, whereas oxamyl had the least value (0.16) in this respect.
Promising results were reported among the tested applications of adding moringa dry leaf powder with calcium sulphate and oxamyl plus potassium silicate at ¼ each dose which showed few number of females (8.5), galls (10.75), egg masses (7) and juveniles (400/1 Kg soil) that can be detected on root system and soil of tomato cv.9065 FI in this study, respectively (Table 2).Data in Table 3 shows the impact of either potassium silicate or calcium sulphate or moringa dry leaf powder alone or mixed as dual or triple or tetra with oxamyl comparing to oxamyl at the recommended dose on nitrogen (N) phosphorus (P), potassium (K), total phenol and total chlorophyll contents in leaves of tomato plant cv.9065F1 infected with M. incognita under greenhouse conditions (25±3C).It was evident that N, P and K concentration were obviously reduced by nematode infection.It is interesting to note that all tested treatments gave remarkable increase in N, P, K and total phenol concentrations exceeding that of nematode alone (Table 3).Among the single treatments, moringa dry leaf powder ranked first in increasing N, P, K and total phenol concentrations with values of 1.64, 0.370, 1.98 and 20.8%, followed by calcium sulphate and then potassium silicate, respectively.
Moreover, an obvious synergetic action occurred in increasing N, P and K concentrations only when oxamyl was added to potassium silicate plus calcium sulphate at ⅓ dose each as triple treatment with values of 2.25, 0.431 and 2.69% and low value of total phenol percent (9.2%), respectively.However, a reasonable concentration of N, P and K was recorded by mixing the three tested components that is potassium silicate, calcium sulphate and moringa dry leaf powder together that exceeded the single treatment at 1/3 dose each with values of 1.93, 0.407 and 2.43% with low value of total phenol (3.4%).It is worthy to note that high synergistic action was evident with tetra application in increasing concentrations of N (2.37%), P (0.446%), K (2.79%) and total phenol (23.7%) that exceeded all treatments in this respect even that the plant free of nematode and any treatment.Moreover, the tetra treatment that contained all items plus oxamyl at 1/4 dose each ranked first in increasing N, P, K and total phenol comparing to nematode alone (Table 3 ).
It is worthy to note that the tetra application [1∕4(calcium sulphate + potassium silicate + moringa dry leaf + oxamyl)], gave the highest percentage increase of total phenol (23.7%) and the highest reduction value of total chlorophyll content (18.1%) comparing to nematode alone (Table 3).
Concerning total chlorophyll content in leaves of tomato plants infected with M. incognita under the tested treatments, the single ones showed the high reduction percentage values that ranged between 14.1 to 16.6% which was more than other double, triple, oxamyl and plant free of nematode and any treatments, whilst the tetra treatment accomplished its highest reduction percentage (18.1%)comparing to nematode alone (Table 3).
Using calcium sulphat or potassium silicate or moringa dry leaf powder either alone or mixed as dual or triple or tetra treatments along with oxamyl at ½ or ⅓ or ¼ doses compared to oxamyl at the recommended dose on reproduction and development of M. incognita play an important role in diminishing root knot nematode on tomato plants and its reproduction factors (RF), where the double, triple and tetra treatments tested gave better results than single ones did.In the meantime, among the single applications, moringa dry leaf powder accomplished the highest percentage reduction of nematode parameters.Results of this work are supported by the findings of Sowley et al. (2014) who said that the infestation of root-knot nematodes were significantly lower in the moringa leaf powder-treated plots than the control.They added although significant differences were not observed in all the parameters evaluated among the moringa leaf powder treatments, sweet pepper plants treated with 80 g/L of moringa leaf powder per plot recorded the highest mean value of plant height, number of leaves, number of fruits per plant, fruit weight per plant total yield per plot and the thickest plant girth.Similarly, the sweet pepper plants treated with 80 g/L of moringa leaf powder had the lowest infection index (root gall) and nematode population.Application of moringa leaf powder at 40, 60 and 80 g/L increased sweet pepper yield and decreased nematode population confirming their potential in management of root-knot nematodes.
Claudius-Cole et al. ( 2010) recorded that Moringa oleifera, is widely used in water treatment as a good inhibitor of nematode egg hatch and juvenile survival.It was also effective in reducing nematode population in plants with a subsequent increase in plant growth and yield.Guzman (1984) found that water extracts of moringa leaves were to be as toxic to M. incognita as standard pesticides.The nematicidal effect of the tested materials may possibly be attributed to their high contents of certain oxygenated compounds which are characterized by their lipophilic properties that enable them to dissolve the cytoplasmic membrane of nematode cells and their functional groups interfering with the enzyme protein structure (Knobloch et al., 1989).
The positive increments values of N, P and K was correlated within any tested single, dual, triple and tetra applications of such materials along with oxamyl added, a situation that is supported by the findings of El-Sherif and Ismail ( 2009) who reported that ½ (B.t.+ Ox) treatment exceeded that of either B.t. or ox amyl alone in values of N, P and K cons. in soybean plant infected with M. incognita.Meanwhile, the same trend occurred in the case of total phenol content on tomato plant infected with M. incognita, in this study and is supported by the findings of Kesba (2010) in respect to treatments of humic and fulvic acids that significantly improved the levels of non-enzymatic antioxidants molecules including total phenol in the roots infected grape rootstocks roots with Rotylenchulus reniformis or Tylenchulus semipenetrans, especially at the higher concentration of the organic acids.Increasing levels of total phenol may serve as defense compounds against pathogens (Kosuge, 1969).However, there were negative correlations between the single and concomitant applications of the tested components regarding the reduction of total chlorophyll content in the present study compared to nematode alone, a condition which is in agreement with those reported by El-Sherif and Ismail (2009) in respect to M. incognita infecting soybean plant.
Undoubtedly, the abiotic factors used in the present investigation as tool for the integrated control of M. incognita on tomato plants through the tested one or two or more components along with oxamyl as systemic nematicide at their proper doses succeeded to generate a sort of inducing resistance in a susceptible host plant against such pathogenic nematode, since tetra application in this work showed very low eggmasses (7.0) or females (8.5) on root system of tomato plant infected with M. incognita.Moreover, the nematicidal activities of the tested materials as biofertilizers as well as their thermo stable toxin in the integrated management of M. incognita on tomato plants along with oxamyl can be varied from component to another.These variations may be attributed to the differences in the chemical nature, compound present in these tested material and method of application used.The safety of such materials and its low cost is one of its advantages.These observations agreed with those of Oteifa (1953) who stated that rootgall nematode damage on cabbage increased with amounts of potassium available to the host plant because potassium increased the rate of reproduction of nematode.Huber (1991) also recorded that root gall nematode damage on lima bean decreased with increased ammonium supplied to the plant.
In addition, these results are also in agreement with those reported by Oteifa and El-Gindi (1962) in respect to plants with fewer root galls that would translocate more nutrients to vegetative organs than heavily galled roots.Regarding the C/N ratio, it ranged between 21.34 to 22.19, 19.83 to 18.26, 16.03 for single, dual, triple and tetra treatments vs. 22.88 for nematode alone, respectively, and agrees with the findings of Miller and Donahue (1990) who reported that organic residues with C/N ratio 20:1 or narrow have sufficient nitrogen to supply the decomposing microorganisms and also to release for plant use.
However, more research is needed to be done in this direction under field conditions before drawing such recommendations for new trend safe and effective integrated nematode management alternative (s) based on the combined use of natural and synthetic compounds.
Acetone volume; D= Optical density reading at the wave length (cm) and D = ∕ cm.

Table 1 .
Impact of calcium sulfate or potassium silicate or moringa dry leaf powder either alone or mixed as double or triple treatments or with oxamyl or as tetra application in comparison with oxamyl on plant growth response of tomato cv.9065 FI infected with 2000 second stage juveniles (J2) of Meloidogyne incognita under greenhouse conditions Values are the mean of four replicates.Ox = oxamyl.*Means in each column followed by the same letter(s) did not differ at p<0.05 according to Duncan's multiple-range test.

Table 2 .
Nematode parameters of Meloidogyne incognita infecting tomato plant cv.9065 F1 as affected by calcium sulphate or potassium silicate or moringa dry leaf powder either alone or mixed as double or triple applications or plus oxamyl as tetra treatments in comparison with oxamyl under greenhouse condition (25 ± 3C).
Values are the mean of four replicates.Ox = Oxamyl.EI= eggmass index RGI= root gall index.*Means in each column followed by the same letter(s) did not differ at p<0.05 according to Duncan's multiple-range test.RF= nematode reproduction factor.

Table 3 .
Nitrogen (N) phosphorus (P), potassium (K), total phenol and total chlorophyll contents in leaves of tomato CV.9065 F1 infected with Meloidogyne incognita treated with calcium sulphate or potassium silicate or moringa dry leaf powder either alone or mixed as double or triple or with oxamyl as tetra treatments in comparison with oxamyl under greenhouse conditions.
Values are the mean of four replicates.Ox = Oxamyl.*Means in each column followed by the same letter(s) did not differ at p<0.05 according to Duncan's multiple-range test.