Meloidogyne incognita population control and nutritional status and productivity of Thompson seedless grapevines managed with different treatments

A two-year field trial was conducted in a vineyard (northern Egypt)cultivated with Thompson seedless grapevines to evaluate the effectiveness of four “alternative” (biological/chemical) treatments, Bacillus megaterium, boric acid, calcium nitrate and chitosan, against the root-knot nematode Meloidogyne incognita (Mi), compared to that of the nematicide oxamyl. The influence of these treatments on plant nutritional status and fruit yield and quality was also assessed. All treatments significantly inhibited Mi reproduction parameters in both seasons, decreasing the numbers of nematode galls and egg masses (roots) and of second-stage juveniles (soil). Oxamyl application resulted in the highest reductions in Mi-reproduction parameters, followed by boric acid, which also showed the highest relative nematicidal efficacy (respect to oxamyl). In the 1st season, the highest fruit yield (10.34 kg/grapevine) was recorded from boric acid-treated plants, followed by that from oxamyl-treated plants (7.50 kg/grapevine); in the subsequent season (2019), oxamyl use led to the highest yield, followed by boric acid + chitosan use (10.04 and 8.62 kg/grapevine, respectively). In both seasons, application of boric acid alone and combined with chitosan enhanced the total soluble solids (TSS)/total acidity ratio in grape juice. All treatments led to higher nutrient contents (leaf petioles) and chlorophyll levels (leaves) as well as enhanced fruit size and weight. We conclude that the tested treatments can be safely applied for nematode management in Thompson seedless grapevines, with positive effects on fruit yield and quality.


Introduction
Grape (Vitis vinifera L.) is one of the most important commercial fruit crops in the world and the first major deciduous fruit crop in Egypt, ranking in the second position after citrus. The
Oxamyl is one of chemical nematicides in Egypt, which was shown to be useful to minimize the impact of PPNs and increase grape [10]. Although chemical nematicides are more effective in reducing nematode-associated yield losses than biological-based methods, in many regions, there are ongoing research efforts to find more environment-friendly strategies to manage nematode infections in economically relevant crops [5,36].
The main objective of this research was to identify alternatives strategies for the effective management of root nematodes in grapevines other than the use of synthetic nematicides. To this purpose, we analyzed the effects of some alternative treatments (including simple minerals, biofertilizers, and bioagents) not only on RKN population but also on the productivity of Thompson seedless grapevines, aiming at producing healthier and less polluted grapes, free of chemical residues, to meet the exportation requirements, with the additional advantage of lower costs.

Site description and vineyard management
The present study was conducted during two successive production seasons (2017/2018 and 2018/2019) in a12-year-old private vineyard located in El-Sahrawi village, El-Beheira Governorate, Egypt. The selected experimental plots included Thompson seedless grapevines growing on sandy soil; they were uniform in growth and vigor and had been planted at a distance of 2.5 × 1.75m in a soil naturally infested with M. incognita. The physical and chemical properties of the experimental soil (0-90 cm depth) were determined and are shown in Table 1.
Grapevines were irrigated with a drip-irrigation system consisting of two lines and four drippers per vine (8 L/h). The grapevines were trained on a three-wire trellis and pruned to 7 canes with 12 eyes along with 7 renewal spurs; in total, 98 buds per vine were left. Standard agricultural practices for commercial grape orchards were carried out. Group 1 (Ct): untreated, positive control. Vines continued growing on the soil naturally infested with M. incognita and received no treatment.

Experimental treatments
Group 2: standard chemical treatment. The nematicide oxamyl (Vydate 24% SL), produced by DuPont company, was applied at the recommended dose (5mL/grapevine). This was the comparative treatment.
A total of 60 vines, as uniform as possible in growth and vigor, were selected and subjected to these treatments, with 5 replicates per treatment and 2 vines per replicate (the number of the treated vines for each applied treatment were 10 vines) (i.e., 6 treatments × 5 replicates × 2 vines per replicate = 60 vines). The experimental treatments were arranged in a randomized complete block design (RCBD), according to Gomez and Gomez [37]. In both tested seasons, each treatment was applied twice, on January 15 and on February15. The treatments were distributed under the canopy of each vine near the stem with 25 cm from each side and then irrigated. Grapevines received the standard agricultural practices according to the recommendation of Ministry of Agriculture, Egypt.

Nematode infection assessment
In both seasons, before the application of the treatments, soil samples were collected with an auger that made a hole of 10 cm in diameter giving a total of about 1000 cm 3 soil from 20 and 30 cm depths from each side (about 25 cm away from the vine trunk). The collected subsamples from each vine were mixed in one compound sample from which a sample was examined to estimate the number of M. incognita second-stage juveniles (J 2 ) per kg of soil using sieving and centrifugal flotation technique [38]; this data was considered the initial nematode population (P i ). In addition, approximately 10 g root samples were collected from each site of the vine to determine the number of root galls (G) and nematode egg masses (EM) using an aqueous solution of phloxin B (0.15 g/L) for 15 minutes to clarify the nematode egg masses [39]. The same parameters were estimated after treatment applications using soil and root samples collected at the end of each season to represent the final nematode population (P f ) and final numbers of nematode galls and egg masses (with consideration that the final population of the 1st season was deemed the initial population of the 2nd season). Species of Meloidogyne incognita was identified based on the perineal pattern of the adult female that dissected from the infected galled roots of grapevine [40]. The perineal pattern characteristics were described according to [41].
The nematode reproduction factor (Rf) was calculated for each treatment by dividing P f /P i [42]. For each nematode population parameter (G, EM, and J2), the percentage of reduction (R) was calculated based on Mulla's formula [43], as follows: Reduction % ðRÞ ¼ 100 À ½ðC1=T1Þ � ðT2=C2Þ � 100�; where: C1 = pre-treatment value in the untreated control (Ct); C2 = post-treatment value in the untreated control (Ct); T1 = pre-treatment value in a given treatment; T2 = post-treatment value in that treatment. To compare the effectiveness of the four alternative treatments to that of the nematicide oxamyl 24%, the relative nematicidal efficacy (RNE) of each one was calculated based on nematode reproduction factors (Rf) and expressed as a percentage, using the following equation:

Grapevine nutrient status and total chlorophyll contents
Two weeks after the fruit set, leaf petioles were collected for mineral composition analysis. The leaves situated opposite to the grape clusters were used to this purpose, as suggested by Cottenie et al. [44]. Samples of 20 petioles per replicate were washed carefully with tap water and distilled water and subsequently dried at 70˚C until constant weight. Dried petioles were ground using an electric mill and digested using sulfuric acid and hydrogen peroxide. Grapevine nutritional status was evaluated by the determination of leaf petiole mineral content [45]. Nitrogen (N) was estimated by the micro-Kjeldahl method [46]; phosphorus (P), by the method of Murphy and Riely [47]; and potassium (K), by flame photometry according to the method of Brown and Lilleland [48]. Calcium (Ca), magnesium (Mg), and iron (Fe) were determined using an atomic absorption spectrophotometer (Perkin Elmer 3300), according to Carter [49]. Boron (B) was colorimetrically determined using the carmine method, as described by Hatcher and Wilcox [50].
Total chlorophyll content (SPAD) was also determined two weeks after fruit set on10 leaves per vine, chosen among those situated from the 6 th or 7 th leaf to the tip of the growing branches, according to the method described by Yadava [51]. A Minolta Chlorophyll METER SPAD-502 was used (Minolta camera, LTD Japan).

Grapevine reproductive performance and yield
The number of burst buds was determined one month after bud burst by simple counting, and the percentage of bud burst, bud fertility, and fruiting coefficient were calculated according to Bessis [52] and Gaser et al. [53], using the following equations: At harvesting time (middle July, both seasons), the fruit yield per vine was estimated by multiplying the number of clusters/vine × average cluster weight.

Fruit parameters of commercial relevance
A total of 30 clusters per treatment (3 clusters/vine) was used to obtain average values for the following physical characteristics: cluster's weight (g), cluster's length (cm), cluster's width (cm), 100-berries weight (g), 100-berries volume (cm3), berry´s length (mm), berry's width (mm). The shape index was calculated as the fruit length/fruit width ratio. Fruit chemical characteristics assessed in the berry juice were the percentage of total soluble solids (TSS) and acidity (TA). The first one was determined using a hand refractometer. Total acidity was measured according to the official methods published by the AOAC [54] and expressed as grams of tartaric acid/100mL of berry juice. The ratio between total soluble solids and total acidity (TSS/ TA) was calculated [55].

Statistical analysis
Treatment effects were evaluated by performing one-way analysis of variance (ANOVA). Means were separated and compared using the least significant difference (LSD) test at 0.05 level of probability, according to Steel and Torrie [56]. The statistical analysis was carried out using the software SAS (Statistical Analysis System), version 9.13 [57].

Nematode infection assessment
Over both experimental seasons (2018 and 2019), all treatments significantly reduced the numbers of nematode galls and egg masses/g fresh root, as well as the numbers of second-stage juveniles (J 2 ) per kg of soil and M. incognita reproduction factors (Rf) ( Table 2 and Fig  1). It may be noticed that the application of boric acid alone was the most effective treatment, resulting in strong nematode populations decreases, without significant differences compared to the reductions observed with the use of the nematicide oxamyl 24%.
The reduction percentages in root-galls numbers (G) by applying the alternative treatments ranged from 50.64 to 80.66% and from 65.37 to 84.54% in 2018 and 2019, respectively. Vineyards treated with the nematicide oxamyl showed 81.96% and 90.31% reductions in this  Table 2). The reproduction factor (Rf) of M. incognita infecting Thompson seedless grapevine was significantly reduced by all treatments (Fig 1). Rf values ranged from 0. 27

Grapevine nutrient and chlorophyll contents
The concentration of several nutrients in the leaf petioles of Thompson seedless grapevine plants and total chlorophyll levels are shown in Table 3. In the 1st season (2018), the application of boric acid alone caused the most significant nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), boron (B), and total chlorophyll (SPAD) rises. In the 2nd season (2019), however, [boric acid + chitosan] was the treatment responsible for the higher increments in leaf total chlorophyll and petiole nutrients except for boron, whose maximum concentration (89.8ppm) was recorded under boric acid application. In both seasons (2018 and 2019), all the other treatments caused significant increases in petiole nutrient levels and total leaf chlorophyll as compared with untreated vines (Ct) except for Oxamyl in the 1st season, which did not significantly elevate petiole potassium concentration (Table 3).

Grapevine reproductive performance and yield
As a general trend, the applied treatments increased bud burst, bud fertility, and fruiting coefficient respect to the untreated control (Fig 3A, 3B and 3C). In the 1st season (2018), the exceptions were B. megaterium, [boric acid +calcium nitrate], and [boric acid + chitosan] treatments, in which the fruiting coefficients did not differ significantly from that observed in control grapevines. Bud burst, bud fertility, and fruiting coefficient reached the highest values in grapevines treated with boric acid: 91.43, 18.57, and 20.31%, respectively. In the 2nd season (2019), all treatments significantly improved the bud burst and bud fertility compared with the untreated control, and the highest bud burst and bud fertility percentages were recorded in oxamyl-treated vines, followed by vines under [boric acid + chitosan] treatment (Fig 3A and  3B). In terms of fruiting coefficient, however, [boric acid + chitosan] treatment was found to be slightly superior to boric acid and oxamyl treatments, being all of them over the control (Fig 3C).

PLOS ONE
Nutritional status and productivity of Thompson seedless grapevines Regarding grape yield, in the 1st season (2018), the highest value was obtained in grapevines treated with boric acid alone (10.34 kg/vine) followed by those which received oxamyl (7.50 kg/vine) (Fig 3D). In the subsequent season (2019), the highest yield was achieved in oxamyltreated plants (10.04 kg/vine), followed by those under [boric acid + chitosan] treatment (8.62 kg/vine) and boric acid treatment (7.79 kg/vine). In both seasons, the lowest yields were recorded in the untreated control (Fig 3D).

Fruit parameters of commercial relevance
The juice of Thompson seedless grapevines harvested during 2018 and 2019 seasons was analyzed. In the 1st season (2018), the highest percentage of total soluble solids (TSS) was recorded in the grape juice obtained from boric acid-treated vines, followed by that obtained from [boric acid + chitosan] treatment ( Fig 4A).
In the 2nd season (2019), conversely, the highest percentage of TSS was detected in [boric acid + chitosan]-treated vines, followed by that measured in the juice from boric acid-treated vines. In addition, both treatments (boric acid and [boric acid + chitosan]) significantly decreased the total acidity (TA) in both seasons (Fig 4B), resulting in significantly higher TSS/ acid ratios (Fig 4C). Treatments with [boric acid + calcium nitrate], B. megaterium, and oxamyl had similar effects on these parameters (less pronounced but still significant compared to untreated plants) in 2018, while in 2019, [boric acid + calcium nitrate] addition led to a significant increase in TA, which resulted in a significant decrease in TSS/acid ratio. The highest TSS/TA ratios corresponded to boric acid-treated vines in 2018 and to [boric acid + chitosan] in 2019. Fig 5 shows the influence of different M. incognita-control treatments on relevant commercial characteristics of the harvested Thompson seedless grapevines. As it may be noticed, in both seasons the treatments applied improved these characteristics including cluster weight (Fig 5A), cluster length (Fig 5B), cluster width (Fig 5C), 100-berry weight (Fig 5D), 100-berry volume (Fig 5H), berry length (Fig 5E), and berry width (Fig 5F). In the 1st season, grapevines treated with boric acid displayed the highest values for these indices, followed by those treated with oxamyl. In the 2nd season, the highest values corresponded to grapevines treated with oxamyl, followed by those subjected to [boric acid + chitosan] treatment. Compared with the shape index (fruit length/fruit diameter width) of the untreated control, all treatments slightly increased this value in the grapes harvested in 2018 (Fig 5G). In those harvested in 2019, boric acid and B. megaterium treatments significantly increased this ratio, while [boric acid + calcium nitrate] treatment significantly reduced it. Treatment with [boric acid + calcium nitrate] generated the lowest increments respect to the untreated control in all physical characteristics assessed, except for the berry width, for which the lowest increment was associated with B. megaterium application (Fig 5F).

Discussion
Based on the assumption that mineral nutrition affects plants' susceptibility to-parasitic nematode diseases, supplying plants with micronutrients may increase plant resistance against nematode pathogens. In this sense, Santana-Gomes et al. [17] stated that balanced plant nutrition could increase plant resistance or tolerance to nematode infection. Plants take up boron through the roots as uncharged boric acid (H 3 BO 3 ) [58]. Although boron is a micronutrient, it was reported that its deficiency affects grapevine growth and fruit set and quality [59] because this element displays a role in fruit sugar accumulation and contributes to pollen germination, pollen tube growth, and normal fruit set [23]. However, boron excess can be toxic to plants affecting their growth [60]. In addition, fruit set clusters tend to be small under boron limitations, and berries cannot fully develop on the rachis [18].
Our results confirm the nematicidal potential of boric acid against the root-knot nematode, and they are in line with those reported by El-Saedy et al. [5], who showed that boric acid application significantly reduced the number of root galls and egg masses of M. incognita on the root system of Thompson seedless grapevines, along with improvements in fruit yield and properties of commercial importance. They also reported the significant nematicidal activity of boron against the citrus nematode, T. semipenetrans, infesting orange trees [24]. El-Saedy et al. [24] informed that boron treatment reduced the final nematode population and had a relative nematicidal efficacy (RNE) of about 83-85%. The application of this micronutrient also improved fruit yield and quality, compared to the untreated control.
While the nematicidal activity of boric acid against plant-parasitic nematodes (PPNs) has not been discussed in the literature, in 2006, Habes et al. [61] elucidated the mechanism involved in the toxicity of this compound to the nervous system of insects. These authors found that boric acid acts as an inhibitor of acetylcholine esterase (AChE) in the German cockroach, Blatella germanica. Since this mode of action coincides with that of many PPNs-targeting commercial nematicides [62], boric acid probably inhibited nematode AChE and thus reduced the nematode population. Surprisingly, boric acid was found to play a dual role in the present study, both as nematicide and fertilizer, and be as effective as oxamyl in controlling M. incognita. Regarding grapevine reproductive performance and yield, our results show that high values were achieved in both seasons when boric acid, alone or in combination with chitosan, was applied. Besides, these treatments led to the highest percentages of total soluble solids (TSS) and the lowest percentages of total acidity (TA), resulting in significantly increased TSS/total acidity ratios. These results are consistent with those of previous reports, which revealed that boron application to vineyards increased grape yield [21,63].
The improvement in grapevine growth following boric acid application may be attributed to nematode population control and probably to boron's involvement in natural phytohormone transport, which may have contributed to both cell division and enlargement [5,64]. Boron has a more critical role in the reproductive period than in the vegetative period [63]. Therefore, in soils with nutritive deficiencies or when plants cannot take up nutrients at the required levels, balanced fertilization is necessary to reach high grape yield [63]. Besides, it was reported that boron addition induces carbohydrates movement and ascorbic acid transfer from plant leaves to the fruiting bodies [21,65], and this effect possibly led to the increase in the fruit weight in our research. Boron's relevance in carbohydrate metabolism [66], cell division, protein synthesis [60], early flower initiation, and flower bud formation [22] was previously documented.
M. incognita-control treatments applied in this study had significant effects on leaf petioles nutrient contents in both tested seasons; they generally increased N, P, Ca, and Mg concentrations. Optimum grapevine growth has been associated with leaf nitrogen and phosphorus concentrations of 1.6-2.8% and 0.2-0.6%, respectively [67]; therefore, we consider that these nutritional requirements were met under the treatments tested in this study. Moreover, calcium and magnesium leaf contents were even above the optimum values proposed (0.4-2.5% and 0.13-0.4%, respectively) [67]. However, leaf potassium concentrations were below the optimum ranges reported by these authors (1.5-5.0%) [67].
The highest nutrient contents (leaf petioles) and total chlorophyll levels (leaves) were recorded for grapevines under boric acid and [boric acid + chitosan] treatments in the 1st and 2nd seasons, respectively. These results are in line with those reported by Gunes et al. [68], who mentioned that both foliar and soil applications of boron increased grapevine yield and the content of N, Ca, Mg, P, K, and Zn in both plant leaves and berry tissues.
Many studies document the nematicidal effect of chitosan against plant-parasitic nematodes, which is considered to depend on the concentration and molecular weight of this polymeric substance [30,32,33,35]. It has been found that chitosan addition reduced root-knot nematode invasion by affecting parasite population parameters (i.e., number of galls, egg masses, females/root system, number of juveniles/250 g soil) and reducing Meloidogyne spp. egg hatching and larval viability [32,34], for which this compound may serve as a natural nematicide. Moreover, chitosan applications were used in various fruits and vegetables to induce plant immunity, thus protecting them from different pests and pathogens [24]. Sharif et al. [29] reported that chitosan had fungicidal, bactericidal, and nematicidal activity.
The exact mode of action of chitosan to control pathogenic nematodes remains unclear, but it was proposed that chitin (the precursor of chitosan) may promote the growth of beneficial chitinolytic microbes that parasitize nematodes eggs [69]. Also, Sharif et al. [29] reported that chitosan treatment regulates the expression of several plant genes involved in plant molecular defense systems such as phytoalexins and pathogenesis-related proteins (PR)).
Our results are in line with those obtained by many researchers, who confirmed the positive effects of chitosan on the growth parameters of several crop plants. In grapevine, chitosantreated plants showed better growth [70,71]. Górnik et al. [72] found that chitosan behaved as a successful grapevine biostimulant, as its application improved the root system development and increased the number of internodes and newly formed canes, as well as their length; also enhanced leaf chlorophyll content.
Likewise, many researchers found that using chitosan as a coating material for fertilizers can control the release rate of the inorganic added nutrients, resulting in the prevention of excessive fertilization and lower production costs. It was also communicated that this practice improves the efficiency of fertilizers' uptake by plants [73,74]. Therefore, chitosan can be used as a biodegradable biofertilizer to avoid the hazards of inorganic fertilizers overuse in horticulture. Chitosan was also found to improve crop yield, and the shelf life of the harvested products, with less environmental contamination [29].
Calcium ion is considered one of the most important plant nutrients affecting plant susceptibility to diseases [75]. According to Hurchanik et al. [28], plants subjected to calcium deficiency are more susceptible to nematodes attack; therefore, soil calcium availability is a key point. Calcium content in plant tissues was related to disease incidence [17]. Pathogens growth and their chances to cause infection increase at higher sugar levels flowing from the cytoplast to the apoplast, a process that occurs mainly at low calcium levels. Besides, calcium ions can significantly inhibit the activity of the extracellular pectolytic enzymes (e.g., galacturonase) produced by various plant pathogens, thus preventing the middle lamella degradation and contributing to cell wall stability [75,76]. It has been found that an increased calcium supply to plants promotes root cell resistance and thus reduces nematodes invasions to roots [75,76].
Calcium can be applied to crops through different mineral fertilizers. For instance, calcium carbide addition reduced the number of galls, egg masses, and juveniles of M. incognita in zucchini, and it led to crop yield increases, which were positively correlated to the application rates [77]. Calcium sulfate application also resulted in a high reduction of M. incognita population density and improved tomato vegetative parameters [76].
Patil et al. [78] measured Ca(NO 3 ) 2 uptake by rice roots infected with the root-knot nematode Meloidogyne graminicola and found that soil nitrogen concentration and predominant form can influence the extent of root invasion by M. graminicola and, consequently, rice yield. These researchers showed that regulating soil nitrate concentration during M. graminicola infection periods can represent a non-chemical method of nematode management in rice crops.
Although grapevine growth requires microelements in small amounts, these nutrients may significantly affect grape properties [26]. It was informed that calcium plays an important role in cell division, as well as in the growth and development of fruit trees [27]. Zhang et al. [79] found that calcium not only promotes plant growth, but it also increases plants' ability to resist diseases and enhances fruit flavor.
In our study, calcium nitrate was applied in combination with boric acid and showed no superiority to other treatments in terms of nematode control or productive grapevine performance. Some previous reviews indicate that the application of multiple elements (calcium, magnesium, iron, manganese, copper, and boron) through fertilizers can increase grape yields and improve fruit properties [80]. Further research using different calcium concentrations and chemical forms is required to shed light on this topic.
Many studies have documented the benefits of adding B. megaterium as arhizosphere-colonizing bacteria owing to its nematicidal activity against several plant parasitic nematodes and proposed its use as a biological control agent in certain crops [23,36,81,82]. In this sense, Khalil et al. [10] found that the final nematode population of Meloidogyne javanica and the productivity of the seedless grapevine cultivar "Flame" were significantly affected by the application of the bioproduct Bio-arc TM under field conditions. Several mechanisms were proposed to explain the biological control exerted by B. megaterium. For instance, Neipp and Becker [81] and Oliveira et al. [83] reported that B. megaterium produces metabolites that probably interfere with nematodes' life cycle. Likewise, Huang et al. [84] reported the release from this microorganism of volatile nematicidal compounds that tend to reduce M. incognita infection.
Other reports, such as those of Padgham and Sikora [82], reveal modifications in plant's exudates caused by the antagonistic bacteria.
According to our results, the application of the bacterium B. megaterium through the bioproduct Bio-arc TM not only contributes to nematodes biocontrol in grapevines but also provides better nutritional status. These findings are in agreement with those reported for rice [85], and sugar beet [13]. Other explanations for the mode of action of this bacterium was previously reported by Radwan [86] and López-Bucio et al. [87], who suggested these bacteria may have the ability to dissolve insoluble phosphorus-containing compounds in soil, enhancing P available levels and thus effectively promoting plant growth, and the induction of auxin and ethylene formation by plants, which may impact on the root system.

Conclusions
Root-knot nematodes cause significant yield losses in many grape-growing regions, and also affect fruit quality. Chemical nematicides play a significant role in minimizing these losses, but they are usually expensive and may have unwarranted effects on human health and cause environmental pollution. Safer alternative control methods to manage this pest should be developed as fast as possible, and this study contributes to that aim. We demonstrated that the application of boric acid alone was as effective as a commercial nematicide in reducing M. incognita burden in the root-soil interface of Thompson seedless grapevines, improving grape yield and fruit quality. Boric acid combined with calcium nitrate or chitosan as well the bioagent B. megaterium also decreased nematode population and enhanced grape yield and quality. Our findings may be useful for the development of more sustainable strategies to cope with nematodes infections in grapevines.