Effect of certain organic amendments and Trichoderma species on the root-knot nematode, Meloidogyne incognita, infecting pea (Pisum sativum L.) plants

Abstract

This study was conducted to evaluate the effects of three fungal species, Trichoderma harzianum (Th), T. viride (Tv), and T. vierns (Tvi), and ground seeds of fennel (Foeniculum vierns) and caraway (Carum carvi), and powdered leaves of basil (Ocimum basilicum) as soil amendments against the root-knot nematode, Meloidogyne incognita on pea root, Pisum sativum under screen house conditions. The treatments were compared to a nematicide, carbofuran 10G, and untreated check. The tested materials significantly (P ≤ 0.05) reduced M. incognita on pea as indicated by the numbers of galls, egg masses, and J₂ per root system in pots, either as single or combined treatments. The combined treatments caused higher average total percentages of nematode reduction than those achieved by single ones. The single treatments recorded the averages total percentages of nematode reduction ranged from 72.4 to 82.0%. Basil waste as single was superior in reducing nematode numbers of J₂ and egg masses in root system per plant, as it achieved the highest average total percentages of nematode reduction (77.9%) than 73. 9 and 72.4% caused by fennel and caraway, respectively. In combined treatments, the average total percentages of nematode reduction ranged from 72.4 to 86.4%. Basil + (Th) caused the highest reduction (86.4%), followed by caraway+Tvi (86.3%) and caraway+Th (86.2%). The nematicide, carbofuran was as effective as most as the treatments in reducing nematode parameters. Also, the treatments highly increased growth and yield criteria of pea infected with M. incognita as indicated by shoot length and fresh and dry weights and root fresh weight, number and fresh and dry weights of pods.

Background

Pea, Pisum sativum L. is one of the most important vegetable crops grown in cool season throughout the world. Peas are rich in starch and high in fiber, protein, vitamin A, vitamin B6, vitamin C, vitamin K, phosphorus, magnesium, copper, iron, zinc, and lutein (Pownall et al., 2010). Among various pathogens attacking pea plants, the root-knot nematode, Meloidogyne incognita, is the most important one that attacks the roots (Anwar and Mcknery, 2010). Since nematicides are very expensive and cause serious environmental hazards, management of nematodes needs to be directed towards the use of natural products. Green manuring of plant parts has been carried out as a method for controlling the root-knot and other plant-parasitic nematodes by several investigators (Youssef and Lashein, 2013; El-Nagdi et al.,2017; Abd El-Khair et al., 2018). Trichoderma spp. are basically mycoparasites (Papavizas, 1985), but in recent years, their suppressive effects against plant nematodes have also been reported (Siddiqui and Shaukat, 2004; Javeed et al., 2016; Abd El-Khair et al.,2019). Soils rich in organic matter are generally colonized by some biocontrol agents such as T. harzianum that improve biocontrol activity. Moringa oleifera when combined with T. harzianum reduced significantly the reproduction, egg hatching, and juvenile numbers of M. javanica on eggplant (Murslain et al., 2014). T. harzianum, when combined with Lantana camara, significantly reduced the root-knot nematode, M. incognita, population criteria on tomato which subsequently highly increased mean of fruit weight and total yield (Feyisa et al., 2015).

The present study aimed to evaluate the efficacy of 3 species, Trichoderma harzianum, T. viride, and T. vierns, with certain medicinal plant wastes alone or in combination to manage the root-knot nematode infecting pea roots.

Materials and methods

Identification and inoculum of M. incognita

Population of Meloidogyne, raised from a single egg mass, was established and collected from eggplant cv. Baladi in a pure culture. Large numbers of egg masses were excised by sterilized forceps from the galled roots. Large numbers of newly hatched 2nd stage juveniles (J₂), used as an inoculum, were prepared by hatching the egg masses after being washed in distilled water, then placed on a sieve layered with a tissue paper at room temperature 23 °C. The sieve was placed over a funnel containing water. This step was repeated several times. The nematode suspension was collected from the plates, and the 2nd (J₂) was placed in a counting dish under stereomicroscope (Askary et al., 2018). Meloidogyne species were identified to be M. incognita, using adult females, on the basis of the morphological and taxonomical characteristics of the female perineal pattern (Barker, 1985).

Preparation of Trichoderma spp. inocula

The inocula of Th, Tv, and Tvi were separately prepared using Sorghum to sand to water (2:2:1 v/v/v) medium. The sterilized medium was individually inoculated by each fungus, using fungal disc (1-cm diameter) obtained from 7-day-old culture. The inoculated medium for each fungal species was incubated at 30 ± 2 °C for 15 days. The resulting fungal inocula were applied in pots’ experiment.

Screen house experiment

Seventy-six pots, each containing 2 kg of solarized sandy loam soil (1:1), were used. The soil was mixed with the tested materials thoroughly to ensure an equal distribution of materials in soil and for further decomposition of plant materials as follows: Trichoderma harzianum (Th), T. viride (Tv), and T. vierns (Tvi) inocula at the rate of 3% of soil weight (w:w) were applied in each pot. Soil in each pot was firstly inoculated in the 10th month of 2018 with Trichoderma spp. and plant materials. Seeds of fennel (Foeniculum vierns) and caraway (Carum carvi) were ground in a blender and added at the rate of 10 g/pot. Also, powdered leaves of basil (Ocimum basilicum) were added at the rate of 10 g/pot. These materials as indicated in Table 1 were used either alone or in combinations against the root-knot nematode, M. incognita, infecting pea roots. The nematicide, carbofuran 10% G (Furadan®) at the rate of 0.02 g/pot (equivalent to 1 kg a.i./feddan = 4200m²), and carrier (Sorghum) were added in each pot under the screen house conditions.

Table 1 Effect of basil, caraway, fennel, Trichoderma harzianum, T. viride, and T. vierns, added alone on second-stage juveniles (J₂), egg masses, and galls of Meloidogyne incognita and number of bacterial nodules in the plant of pea root system under pot conditions

Fifteen days later, pea cv. Concessa seeds were sown in each pot at the rate of 2 seeds per pot. In the 11th month of 2018, each pot was inoculated by 1000 newly hatched J₂ of M. incognita (in 4 holes made around the plant). The pots were watered when needed and were divided into 2 groups: (1) botanical treatments and (2) fungal treatments in single treatments. In combined treatments, they were divided into three groups according to their combinations. All pots were arranged according to a completely randomized design on a bench in the screen house with 5 replicates. Equal number of replicates was served as untreated check. All pots were inoculated with okadean (containing nitrogen-fixing bacterium namely, Rhizobium leguminosarum) at recommended rate.

Procedures of counting nematodes

Three months after inoculation, root system in each pot was cut into two halves. Numbers of egg masses as well as the number of galls were counted under binuclear in one half of roots. The numbers of J₂ in the 2nd half of roots were cut into small pieces. Then, J₂s were extracted by incubation method (Young, 1954) in tap water to help egg hatching from egg masses. Also, numbers of bacterial nodules were counted. Average total percentages’ reduction or increase was calculated by dividing sum percentages of nematode reduction, plant growth, and yield increases of all parameters of each treatment/number of these parameters. This measurement was used to compare treatments within all groups. Also, average overall percentages reduction of nematode, plant growth, and yield increases were calculated by dividing sum average percentages reduction or increase of all treatments of all criteria in each group/number of parameters to compare among different groups.

Plant growth and yield parameters

The plant growth and yield parameters were recorded after 3 months of nematode’s inoculation.

Interaction of data for mixtures

This was estimated for the average total percentages of nematode reduction according to Lempel’s formula reported by Richer (1987) as follows:

$$ \mathrm{E}=\left(\mathrm{X}+\mathrm{Y}\right)-\mathrm{XY}/100 $$

where E = the expected effect of the mixture.

X = the effect due to component A alone.

Y = the effect due to component B alone.

The expected effect was compared to the actual one obtained experimentally from the mixture to determine the additive or synergistic and antagonistic effect according to the equation given by Mansour et al. (1966) as follows:

$$ \frac{\mathrm{Co}-\mathrm{toxicity}=\mathrm{Observed}\ \mathrm{effect}\left(\%\right)-\mathrm{Expected}\ \mathrm{effect}\left(\%\right)\times 100}{\mathrm{Expected}\ \mathrm{effect}\ \left(\%\right)} $$

This factor was used to classify the results into 3 categories where a positive factor 20 or more was considered potentiation, a negative factor − 20 or more was considered antagonistic, and immediate values between − 20 and + 20 indicate additive.

Statistical analysis

Data were statistically analyzed on the basis analysis of variance (ANOVA) procedures. Duncan’s multiple range test as reported by Snedecor and Cochran (1989) was applied for detecting variations among treatments at 5% level of probability. This was done by Computer Statistical Package (COSTAT) User Manual Version 3.03, Barkley Co.

Results and discussion

M. incognita-related parameters

Regarding nematode parameters, 2 measurements were used to compare among treatments and their groups. The first measurement was the average total percentages’ reduction of nematode parameters among treatments of the tested materials within all groups. The second was the average overall percentages’ reduction of nematode criteria among different groups of treatments. Results showed that T. harzianum (Th), T. viride (Tv), or T. vierns (Tvi) and amended with basil, caraway, or fennel plant wastes significantly (P ≤ 0.05) reduced M. incognita. The numbers of galls, egg masses, and J₂ on the root system of pea were less than untreated check (Tables 1, 2, 3, 4, 5, 6, 7, 8, and 9).

Table 2 % Effect of Trichoderma harzianum, T. viride, T. vierns, basil, caraway, and fennel, added alone on the root-knot nematode, Meloidogyne incognita, and number of nodules in pea root under pot conditions

Table 3 Effect of basil, caraway, and fennel, Trichoderma harzianum, T. viride, and T. vierns, added alone on vegetative growth parameters of pea root infected by Meloidogyne incognita

Table 4 Effect of basil, caraway, fennel, Trichoderma harzianum, T. viride, and T. vierns on pod parameters of pea root infected by Meloidogyne incognita

Table 5 Effect of basil, caraway, and fennel, added in combination with Trichoderma harzianum, T. viride, and T. vierns on Meloidogyne incognita and number of nodules in pea root system

Table 6 Effect of combined addition of basil, caraway, and fennel, each with Trichoderma harzianum, T. viride, and T. vierns on Meloidogyne incognita and number of nodules in pea root system

Table 7 Type of interactions of basil, caraway, and fennel each combined with Trichoderma harzianum, T. viride, and T. vierns on the root-knot nematode, Meloidogyne incognita, infecting pea root system

Table 8 Combined effect of basil, caraway, and fennel each with Trichoderma harzianum, T. viride, and T. vierns on certain vegetative growth parameters of pea infected by Meloidogyne incognita

Table 9 Effect of basil, caraway, and fennel, each combined with Trichoderma harzianum, T. viride, and T. vierns, on pod parameters of pea root infected by Meloidogyne incognita

In single treatments, the average total percentages of nematode reduction per root system of pea ranged from 72.4 to 82.0% (Tables 1 and 2), while in combined treatments, they ranged from 72.4 to 86.4% (Tables 5 and 6). The combined treatments caused higher average total percentages of nematode reduction than those achieved by single treatments. It is worthy to notice the basil waste, as soil amendment was superior to those of caraway and fennel in reducing nematode numbers of J₂ and egg masses in roots, as it achieved the highest reduction (77.9%) than (73.9 and 72.4%) caused by fennel and caraway, respectively. Reduction in the number of galls followed the same trend. Also, T. vierns was superior in reducing nematode numbers, as it recorded the highest reduction (82.0%) compared to 78.3% caused by basil + other 2 Trichoderma spp. The recorded percentages of reduction in the number of galls followed the same trend.

Using the average overall percentage, fungal treatments in one group recorded high averages overall percentages nematode and gall reductions (79.5 and 74.1%) and were higher than those recorded by the botanical treatments group (74.7 and 66.7%), respectively. Vice versa, the number of nodules behaved as an independent pattern as botanical treatments caused higher average overall percentages of increase (22.9%) than fungal treatments (17.2%) (Table 2).

The combined treatment of basil + Th caused the highest average total percentages’ nematode reduction (86.4%), followed by caraway + Tvi (86.3%) and caraway + Th (86.2%) than the other combined treatments. By comparing different groups, using average overall percentage, the combined treatments of basil + fungal treatments in one group recorded the highest average overall percentage of nematode reduction (84.1%) than 83.9 and 79.1% recorded by caraway or fennel + fungal treatments, respectively. Also, the reduction in number of galls recorded the same trend as the highest average overall percentages of gall reduction (79.0%) was caused by the group of basil + fungal treatments than the other botanicals + fungal treatments. The percentages of increase in the number of nodules were nearly equal at different combined treatments, based on average overall percentages of the number of nodule increases. Then, nematicide, carbofuran, was as effective as most of the treatments in reducing the number of J₂ in roots and egg masses (75.9%) and root galling (70.4%) and recorded the highest percentage increase in number of nodules (45.7%) than the other single and combined treatments, except that caused by basil treatments. Sorghum carrier recorded the least average total percentages of nematode reduction (70.2%) (Table 6).

Co-toxicity factors for the 2 applied combined treatments of basil, caraway, or fennel, each with the 3 fungal species added at the same time, showed additive or synergistic interaction effects for the average total percentages nematode reduction on pea (Table 7). Co-toxicity for the applied combined treatments showed this additive interaction effects reflecting on the highest percentages of nematode reduction on pea. Similarly, Radwan et al. (2004) indicated that combination between Bacillus thuringiensis and poultry manure, sawdust, grape marc, and chicken litter exhibited additive effect on M. incognita on tomato. Farrag (2011) reported that the combination of Bacillus megaterium, Trichoderma album, and Ascophyllum nodosum each with chamomile, chitosan, or oxamyl showed an additive effect in most cases against M. incognita on tomato. Youssef et al. (2015) found that P. fluorescens + a commercial product named phosphorine containing B. megaterium caused an additive effect in controlling M. incognita on green bean under field conditions.

Plant growth-related parameters

Regarding plant growth parameters, 2 measurements were used for comparison among treatments. The first measurement was average total percentages of plant growth parameter increases among treatments within all groups. The second was average overall percentages of plant growth criteria among different groups of treatments. On this basis, the pots treated with Th, Tv, or Tvi, and basil, caraway, or fennel plant wastes significantly (P ≤ 0.05) increased length and fresh and dry weights of shoots and root fresh weight of pea infected with M. incognita than the untreated check(Tables 3 and 8).

In the single treatments, it is worthy to notice that caraway waste was superior to other plant wastes as it recorded the highest average total percentages of plant growth increase (38.4%) than basil or fennel and untreated check. Also, Th was superior to other fungal species depending upon average total percentages of plant growth increase (Table 3). The 3 Trichoderma spp. in one group caused higher average overall percentages of plant growth increases (46.1%) than that caused by another group of plant wastes (33.5%) (Table 3).

In the combined treatments, basil, caraway, or fennel + Th caused higher average total percentages of plant growth increases than the other treatments and untreated check. Basil waste + each of the 3 Trichoderma spp. in one group caused the highest plant growth increase (89.1%), followed by other groups of fennel waste + each of the 3 Trichoderma spp. (89.0%) and caraway waste + each of the 3 Trichoderma spp.(77.4%). Carbofuran registered intermediate value (61.3%). Sorghum recorded the least one (Table 8).

Yield (pod)-related parameters

As to pod parameters, the same 2 measurements mentioned above related to yield criteria were used for comparison among treatments and their groups. On this basis, the pots treated with Th, Tv, or Tvi, basil, caraway or fennel plant wastes significantly (P ≤ 0.05) increased number and fresh and dry weights of pods of pea infected with M. incognita than the untreated check (Tables 4 and 9).

In single treatments, caraway waste recorded higher average total percentages of pod parameters increase (212.0%) more than those caused by other botanical treatments. The fungus T. harzianum (Th) recorded a higher pod parameter increase (62.3%), than the other fungal treatments. Also, botanical wastes recorded a higher average pod parameters increase of 103.5% than the fungal treatments (49.4%) and untreated check (Table 4).

In combined treatments, the basil + Th, caraway + Tvi, and fennel + Tv caused higher percentages of pod parameter increase (229.8, 301.9, and 211.8%), respectively than other treatments and untreated check. When basil was combined with each of the 3 Trichoderma spp. in one group, it increased average overall percentages of pod parameters by (230.7%) higher than the groups of caraway or fennel + each of the 3 Trichoderma spp. and untreated check. Carbofuran registered intermediate value (44.9%) (Table 9).

Obtained results showed that some medicinal plant wastes had nematicidal effect against M. incognita parameters viz., J₂ in roots; galls and egg-masses in pea. In the other words, the tested powdered leaves and seeds, when used as soil amendments, caused considerable reduction of the root-knot nematode, M. incognita, infecting pea, and subsequently increased plant growth and yield criteria. These results agree with those obtained by Youssef et al. (2004), Youssef and Lashein (2013), and El-Nagdi et al. (2017). The exact mechanism of action of organic amendments may be due to that secondary products from their decomposition are directly toxic to nematodes (Mahmood and Saxena, 1992). Besides, it is possible that nematicidal activity, at least by nitrogenous by-products, should be the most evident when the C:N ratio of the amendment is less than 20:1 (Stirling, 1991). On this basis, basil waste as soil amendment was superior to those of caraway and fennel in reducing nematode numbers of J₂ and egg masses in roots. Montasser (1991) concluded that tannins may be responsible for the suppressive effect on M. arenaria and M. javanica. Also, essential oils have the ability to reduce certain phytonematodes (Abd-Elgawad and Omer, 1995).

Also, the present results showed that the Tv, Th, or Tvr, combined with basil, caraway, and fennel wastes had the highest nematicidal effect against the same M. incognita parameters. These results agree with those recorded by Olabiyi and Gbadamosi (2013) who stated that T. harzanium combined with composted materials reduced nematode population and gall index in sesame. T. vierns combined with the plant debris of oak forests also significantly decreased the number of galls of M. javanica on tomato roots (Moradi et al., 2015). Greater sporulation and multiplication of T. harzianum were supported by decomposed leaves of neem as a medicinal plant as reported by Khan et al. (2012). Direct parasitism of T. harzianum on M. javanica in vitro was reported (Sharon et al., 2001). They showed that protease enzyme and chitinase were secreted by the fungus, Trichoderma, in the culture filtrate that may inhibit egg hatch. Nematicides provide very effective and faster results by reducing nematode numbers and reproduction rates, but they are not environmentally friendly and have many drawbacks (Abd-El-Khair et al., 2019).

Conclusions

Combining Th, Tv, and Tvi fungi with basil, caraway, and fennel wastes provided satisfactory control of the root-knot nematode in pea. The integrated treatments of plant wastes and biocontrol agents not only did reduce suppressive effect of the nematodes, but also caused an additional increase in growth and pod production criteria of pea. The tested fungal species were isolated from Egyptian soil, and medicinal plants are commonly cultivated in Egypt and their leaves and seeds can be further collected and applied with Trichoderma spp. in the field for better root-knot nematode control in pea, improving soil fertility and subsequently, increasing plant growth and yield.