Pathological effects of Meloidogyne incognita eggs on growth of sugarbeet and nematode reproduction under greenhouse conditions

The impact of three levels of Meloidogyne incognita eggs, that is, 1000, 2000 and 3000 eggs/ plant on plant growth parameters of sugarbeet cv. Nejma and its development and reproduction under greenhouse conditions at 17 ± 5°C was studied. Results indicated that reduction percentage of sugarbeet growth parameters were greatly affected, where the marginal effect was more pronounced on roots than shoots. Moreover, as the inoculation level increased from 1000 up to 3000 eggs of M. incognita /plant, the percentage reduction of plant growth parameters increased, where the highest values were recorded to be 48.97 and 24.18% by 3000 eggs/ plant for shoot dry and whole plant fresh weights, whereas, their lowest values of 30.93 and 4.05% resulted by inoculation level of 1000 eggs/ plant for the same plant growth criteria, respectively. Moreover, the highest rate of nematode reproduction on sugarbeet plants cv. Nejma was recorded by the level of 2000 eggs per plant with value of 1.05 whereas the lowest ones of the same criterion resulted by the levels of 3000 or 1000 eggs per plant that were 0.85 or 0.84, respectively. M. incognita infection to sugar beet plants cv. Nejma at the three levels of egg inoculation obviously reduced N, P, K and total chlorophyll content on leaves of sugarbeet with values of 9.9% (N), 18.5% (P), 8.8% (K), 1.58% (chlorophyll) and 20.0% (N), 27.4% (P), 18.4% (K) and 5.9% (chlorophyll) by 1000 and 3000 eggs/ plant, respectively compared to the uninoculated plant.


INTRODUCTION
Sugarbeet (Beta vulgaris L.) is an important arable crop, traditionally used for sugar extraction all over the world.Its total cultivated area reached 227.856 thousands feddans with an average 17.94 tons /Feddan (Feddan = 4200 m 2 ) of sugar beet tubers in the season of 2008/ 2009 (Annual technical report of the council of sugar crops, Ministry of Agriculture, 2005Agriculture, -2009) ) in Egypt, where it is grown in all types of soil especially, in newly reclaimed sand areas such as El-Hamoal Barrary, West Nubaruia, and Al-Bostan regions.Sugarbeet plants are subjected to be attacked by several plant parasitic nematodes in many countries.In recent years, sugar beet is becoming an important crop in Egypt for supporting the expansion of Egyptian sugar industry, therefore efforts to protect the crop from the most destructive pests, that is, nematode and diseases are crucial.In 1976, Jatala and Jensen (1976) described that the interrelationships of Meloidogyne hapla and Heterodera schachtii on sugarbeet in combinations of several inoculum levels given at different times were studied.The numbers of M. *Corresponding author.E-mail: elsherifmohammed@yahoo.com.hapla galls were fewer in any treatment in which H. schachtii was inoculated 10 days before M. hapla than when M. hapla was inoculated first or alone.Noninoculated controls generally had higher concentrations of K, P, Mg, and B than inoculated plants.Pathak and Keshari (2000) showed the effect of inoculum levels of M. incognita on seed germination, seedling emergence, and growth of beet root (Beta vulgaris var.crassa cv.Crimson Globe) in Bihar, India.They stated a significant reduction in seed germination was obtained at 500 nematodes/kg soil, with the reduction being higher in unsterilized soil.Seedling emergence was significantly reduced by at least 1000 nematodes/kg soil in steam-sterilized soil, while the reduction decreased with increasing levels of nematode population in unsterilized soil.Growth significantly decreased with 50 nematodes/kg soil, and continued to decrease with increasing levels of the nematode in the soil.Therefore, the aim of the present work was to deal with the impact of M. incognita at three levels of eggs on growth of sugarbeet and nematode reproduction under greenhouse conditions at 17 ± 5°C.

Nematode source (Inoculum)
To collect and determine the inocula of Meloidogyne incognita eggs, M. incognita was identified according to Taylor et al. (1955).Infected root systems with heavy egg masses of M. incognita various growing coleus plants grown in 25 cm plastic pots filled with sterilized loamy sandy soil at the Nematology Research Unit, Agriculyure, Zoology Department, Faculty of Agriculture, Mansoura University, Egypt, were well-washed and cleaned by running tap water, then placed in a plastic container with enough solution of 1.0% NaOCl for 90 s, shacked vigorously (manually) then, quickly NaOCl solution was passed through 60-mesh sieve, nested over a 400-mesh sieve to collect free eggs.After that, the 400-mesh sieve with eggs was quickly placed under a stream of tap water for several minutes to remove residual NaOCL.Eventually, the number of eggs per unit volume of water was counted and then the seedlings were inoculated directly with eggs levels (Hussey and Barker, 1973).

Pathogenicity test
Sixteen plastic bags were filled with 3 kg/bag of steam-sterilized clay-sand soil (1:1) (V:V) (Table 2).Three seeds of sugarbeet (Beta vulgaris cv.Nejma) were sown planted in each bag.Thirty days from seeds germination and thinning to one seedling / bag, the tested three levels of M. incognita eggs, that is, 1000, 2000 and 3000 eggs were separately monitored.Each inoculated treatment was replicated four times and another four bags left without nematodes served as control.All bags were arranged in a randomized complete block design under the greenhouse conditions at 17 ± 5°C.Plants were watered and regularly receiving conventional pesticides to control mites and insects as needed.After 60 days from the inoculation time, plants were harvested.Plant growth criteria, that is,.shoot and root lengths and fresh weights, as well as shoot dry weight were recorded.
Number of M. incognita second stage juveniles (J2) in 250 g soil/bag was extracted by sieving and modified Baermanntechnique (Goodey, 1957), then calculated for the soil of each bag counted by Hawksely counting slide under 10×4 magnification and recorded.Infected roots of each plant were washed with tap water, then fixed in 4% formalin for 24 h and stained with lactic acidfuchsin 1% (Byrd et al., 1983) and then examined for the number of galls, developmental stages, females and egg-masses.
Data were subjected to analysis of variance (ANOVA) according to Gomez and Gomez (1984), followed by Duncan's multiple range tests to compare means (Duncan, 1955).

Effect of inoculum levels of M. incognita eggs on sugarbeet growth
Data in Table 1 on growth parameters of sugarbeet cv.Nejma revealed that the tested inoculums levels of M. incognita eggs reduced the plant growth as compared to the un-inoculated plants.It is interesting to note that the marginal effect was more pronounced on roots than shoots.Significant differences could be detected among the inoculation levels and the control in total plant fresh weight except that of 1000 eggs level.The same trend was noticed with total plant length.The highest reduction percentage of shoot dry and whole plant fresh weights was achieved by 3000 eggs/bag (plant) since their values amounted to 48.97 and 24.18%, respectively, however their least values resulted by inoculation level of 1000 eggs per plant which amounted to 30.93 and 4.05%, respectively (Table 1).These findings are in accordance with those reported by Gohar and Maareg (2009) who mentioned that gall index was found to be the most sensitive method for assessing resistance of such sugarbeet varieties to M. incognita infection, especially at the low inoculum levels (500 and 1000 eggs) per plant.

Effect of different inoculum levels of M. incognita eggs on its development and reproduction
Table 2 showed that the rate of nematode build-up, number of galls, developmental stages, females and eggmasses were positively affected by the tested levels of eggs.The highest rate of nematode reproduction on sugarbeet plants was recorded at using the level of 2000 eggs per bag with value of 1.05.However, the lowest values for rate of nematode reproduction resulted by either 3000 or 1000 eggs per bag that were recorded to be 0.82 and 0.84, respectively.It was also evident that as the level of M. incognita eggs increased, number of nematode galls and egg-masses increased with root gall and egg-masses indices values of 3.0 and 3.0, 5.0 and 5.0, and 5.0 and 5.0 for 1000, 2000 and 3000 eggs inoculum levels, respectively.Regression analysis of nematode build-up reached the maximum value of R2 that was amounted to 0.5827 (Figure 1).These findings of the present investigation are in accordance with those reported by Osman and Kheir (1978) who reported that growth of sugarbeet cv.Detroit decreased and root-knot     (Taylor and Sasser, 1978).index increased with increasing inoculum levels from 1000 up to 8000 j 2 where maximum reduction in plant growth occurring with 4000 j 2 of M. incognita per plant, and that of Pathak and Keshari (2000) in respect to growth of sugarbeet var.crassa cv.crimson which was decreased significantly with 50 nematodes of M. incognita per kg soil and continued to decreased with increasing volume of nematodes in the soil.Each value is a mean of four replicates.Mean values in each column followed by the same latter(s) did not differ  Also, data presented in Table 3 show the impact of M. incognita infection to sugarbeet plants cv.Nejma at the tested levels of eggs on the content of N, P, K and total chlorophyll in leaves.It was evident that nitrogen, phosphorus, potassium and total chlorophyll content of sugar beet plants cv.Nejama were obviously reduced by nematode infection at the three levels of eggs.Moreover, it was also noticed that as the eggs inoculum level increased from 1000 eggs up to 3000 eggs / plant as these tested elements gradually decreased, since their values were 9.9 (N), 18.5 (P), 8.8 (K), 1.58% (chlorophyll), and 20.0 (N), 27.4 (P), 18.4 (K), 5.9 (chlorophyll) for 1000 and 3000 eggs as nematode inoculation per plant, comparing to the uninoculated plant, respectively (Table 3).Undoubtedly, more work is needed in this respect under field condition before recommendations can be made for management programmes of root-knot nematodes.

*
Each value is a mean of four replicates.Mean values in each column followed by the same letter(s) did not differ at P< 0.05 according to Duncan's multiple-range test..

Figure 1 .
Figure 1.Regression (R 2) value of Meloidogyne incognita nematode build-up rate as affected by the three levels of eggs infecting sugarbeet cv.Nejma under greenhouse conditions at 17±5°C.

Table 1 .
Plant growth of sugarbeet cv.Nejma as affected by three inoculum levels of M. incognita eggs infection under greenhouse conditions at 17± 5ºC.

Table 2 .
Development and reproduction of M. incognita infecting sugarbeet cv.Nejma as influenced by different inoculums levels of eggs under greenhouse conditions at 17 ± 5°C.

Table 3 .
Influence of three inoculum levels of Meloidogyne incognita eggs infecting sugarbeet cv.Nejma on the leaves content of N, P, K, and chlorophyll A, B under greenhouse conditions at 17± 5ºC.