Meloidogyne javanica parasitism on the vegetative growth and nutritional quality of carrots

: Meloidogyne javanica is a plant-parasitic nematode that infects a wide range of vegetables. Its negative effects on crop yield and value are well documented. However, few studies have investigated the impact of the parasite on the nutritional value of vegetables. This study aimed to assess the effect of M. javanica parasitism on the vegetative characteristics, nematological parameters, chemistry composition and antioxidant activity of carrots. Seedlings were inoculated with 0 (control), 1000, 2500, or 5000 eggs and eventual second-stage juveniles (J2) of M. javanica. At 60 days after inoculation, plants were harvested and evaluated. Plants inoculated with 2500 eggs and J2 of M. javanica had higher root and tuber fresh weight than the control. Gall number increased with increasing inoculum density. The number of nematodes in the roots increased until 3000 specimens, decreasing thereafter. Proximate analysis revealed that plants inoculated with 1000 eggs and J2 of M. javanica or more had higher protein content in roots. In contrast, inoculation with 1775 nematodes or more resulted in a decrease in carotenoid content. There was no effect of inoculation on total phenolic content or antioxidant activity. Although, M. javanica infection did not have a marked impact on the nutritional quality of carrots, gall formation resulted in deformed roots of low commercial value.

Root-knot nematodes, such as Meloidogyne javanica and M. incognita, are one of the major limiting factors to carrot crop yield, leading, in some cases, to total production loss (COLLANGE et al., 2011;VIGGIANO et al., 2014). During feeding, nematodes inject esophageal secretions into plant tissues, causing hypertrophy and hyperplasia of cortical cells of the host root. This process leads to the formation of root galls, which alter root shape, affect water and nutrient transport, and; consequently, reduce vegetative growth (HUSSAIN et al., 2016). Changes caused by root-knot nematode infection, even when at low levels, are responsible for reduced commercial value (GUGINO et al., 2006). Affected carrots; albeit being unsuitable for the consumer market, can be used in the food industry for compound extraction or processing. However, studies on tomato and bean have shown that stress caused by nematodes can alter the chemical and nutritional composition of vegetables (AHMED et al., 2009;ATKINSON et al., 2011).
Research on the effects of nematodes on carrot plants is limited to susceptibility analyses.
There is little information about the influence of such parasitism on the proximate composition and nutritional quality of the vegetable. This study aimed to fill this research gap by assessing and quantifying the nutritional loss and physical damage caused by root-knot nematode infection in carrots and presenting alternative uses for affected roots.

MATERIALS AND METHODS
The experiment was conducted in a greenhouse using a completely randomized design with four treatments, four replications for vegetative parameters and nematode population density, and three replications for proximate composition and antioxidant activity. Treatments consisted of the following inoculum levels: 0 (T1, control), 1000 (T2), 2500 (T3), and 5000 (T4) eggs and eventual second-stage juveniles (J2) of M. javanica plant −1 .
Thirty days after planting, carrot seedlings were thinned to one per pot and inoculated with 4 mL of a suspension containing different levels of M. javanica eggs and second-stage juveniles (J2). Eggs and J2 were extracted according to the method of HUSSEY & BARKER (1973) adapted by BONETI & FERRAZ (1981) and counted using a Peters chamber under a light microscope (BA210 Binocular, Motic, Hong Kong, China). Irrigation was performed daily, and foliar fertilization was conducted every 15 days using 5 g L −1 Nutrijá ® (380 g of N, 380 g of P 2 O 5 , and 380 g of K 2 O; Agrária, Jardinópolis, Brazil).
Plants were collected 60 days after inoculation, and the material was separated into shoots and roots (tuber + secondary roots). The following vegetative parameters were assessed: shoot fresh weight, shoot dry weight, shoot height, root fresh weight, tuber fresh weight, and tuber length. Shoot height was measured with a millimeter ruler and tuber length was measured using a Pantec ® digital caliper. Shoot dry weight was determined after drying in a forced air-oven at 65 °C for 3 days.
Nematodes were extracted from roots and tubers following the method of CHARCHAR et al. (2006), with modifications. Roots and tubers were peeled to a depth of about 3 mm, cut into 1-2 cm pieces, and homogenized with 0.5% hypochlorite solution in a blender for 30 s at high speed. The suspension was sieved through 60-(0.250 mm) and 500-mesh (0.025 mm) sieves. The material retained in the bottom sieve was washed with water to obtain the nematode extract. Eggs and J2 were counted using a Peters counting chamber under a light microscope (BA210 Binocular, Motic, Hong Kong, China). The total number of nematodes was divided by the root fresh weight to obtain the nematode population density (nematodes g −1 root). The reproduction factor (RF) was calculated as the ratio of total population to inoculum level (OOSTENBRINK, 1966 Carotenoids were extracted by maceration of 3 g of freshly harvested carrots with 30 mL of acetone. The mixture was filtered, and the liquid fraction was transferred to a separatory funnel containing 60 mL of petroleum ether. The organic phase was washed three times with 300 mL of distilled water to remove the acetone and used as carotenoid extract. Absorbance was read spectrophotometrically (700Plus, Femto, São Paulo, Brazil) at 453 nm. Carotenoid content (C, µg g −1 ) was calculated as follows: C = A × V × 10 4 ÷ E × W, where A is the absorbance, V is the total volume of carotenoid extract, E is the extinction coefficient of β-carotene in petroleum ether, and W is the weight of sample used to prepare the extract (RODRIGUEZ et al., 1976).
Antioxidant compounds were extracted according to RAVICHANDRAN et al. (2013), with minor modifications. Fresh samples were dissolved in 20 mL of 50% ethanol, stirred for 4 h on an orbital shaker, and centrifuged (MTD III plus, Metroterm, Porto Alegre, Brazil) at 3000 rpm for 10 min. The supernatant was collected, and the extraction process was repeated twice more with 5 mL of 50% ethanol. The ethanolic extract was used for measuring total phenolic content (TPC) and antioxidant activity.
Data were subjected to analysis of variance and regression analysis at the 5% and 10% significance levels using the Sisvar software version 5.6. Nematode and vegetative growth data were transformed to to meet normality assumptions based on the Shapiro-Wilk test. Pearson correlation analysis was performed at the 5% significance level using Statistica version 8.

RESULTS AND DISCUSSION
Gall number increased with increasing inoculum density (Figure 1a), as also observed in parsley and spring onion exposed to 0, 760, and 5700 M. incognita eggs + J2 (WALKER, 2002) and cucumber inoculated with M. incognita (KAYANI et al., 2017). The number of nematodes in the roots increased with inoculum density up to 2500 nematodes plant -1 but decreased thereafter ( Figure  1b). Carrot plants were susceptible to all inoculum densities (RF > 1), but multiplication was affected by high initial population densities. At an inoculum density of 1000 nematodes plant −1 , the RF was 11.47, whereas at 5000 nematodes plant -1 , the RF was reduced to 1.57 (Figure 1b). This result is likely due to competition between nematodes, as previously observed in other plant pathosystems (HUSSAIN et al., 2016). No differences (P > 0.10) were observed between treatments in nematode density, ranging from 1133 to 1726 nematodes g −1 root.
Shoot dry weight (2.21-2.85 g) and tuber length (10.97-12.15 cm) were not affected by M. javanica inoculum levels. Conversely, shoot height, shoot fresh weight, root fresh weight, and tuber fresh weight increased (P < 0.05) up to inoculum densities of 4000, 2700, 2125, and 2250 nematodes plant -1 , respectively ( Figure 2). Although, nematodes stunt the growth of the root system by inducing necrotic lesions and galls (AFSHAR et al., 2014;HUNT & HANDOO, 2009), root and tuber weight can increase with gall formation (KAYANI et al., 2017). Galls form from the hypertrophy and hyperplasia of vascular parenchyma in root cells affected by secretions of the dorsal esophageal glands of nematodes. Root galls hamper the formation of secondary roots, reduce water and nutrient absorption, and impair plant growth (ESCOBAR et al., 2015). It is possible that the 60-day experimental period was not long enough for the effects of M. javanica parasitism on shoot development to become evident. Moreover, plants may still develop when exposed to small nematode populations, as new roots can emerge (OLTHOF & POTTER, 1977). This could explain the high root fresh weight and tuber fresh weight of plants inoculated with up to 2125 and 2250 nematodes, respectively, compared with plants inoculated with higher nematode densities (Figure 2c and 2d).
Moisture and ash contents did not differ significantly between treatments, ranging from 852.36 to 853.86 g kg −1 and from 11.40 to 11.86 g kg −1 in T1 and T4, respectively. Protein levels increased with inoculum densities greater than 1000 nematodes plant -1 (Figure 3a). This result can be attributed to the plant's response to stress caused by nematode Débia et al. infection (ROCHA et al., 2015). Plants have a wide array of mechanisms to respond to biotic and abiotic stresses (ATKINSON et al., 2011), including metabolic activation, phytoalexin biosynthesis, phenolic compound accumulation, and increase in peroxidase, catalase, phenylalanine ammonia-lyase, and β-1,3-glucanase activities (DIAS-ARIEIRA et al., 2013). Thus; although the current study assessed the level of total proteins rather than that of pathogenesis-related proteins, it is likely that proteinrelated defense mechanisms were activated by high inoculum densities. A previous study showed that nematodes parasitism can alter the protein profile of plants (GHEYSEN & FENOLL, 2002). These effects should be further studied.
Carotenoid content increased up to inoculum densities of 1775 nematodes plant -1 , decreasing thereafter (Figure 3b). A similar effect was observed in mung bean (Vigna radiata) inoculated with 2000 M. javanica eggs + J2. The reduction in carotenoid content was attributed to a reduction in leaf area (AHMED et al., 2009). Oxidation is the main cause of carotenoid degradation. The compound is easily oxidized because of its large number of double bonds. Carotenoids are protected from oxidation in intact tissues; however, tissue damage increases susceptibility to oxidation (SAINI et al., 2015). Activation of plant defense responses to nematode infection probably increased the activity of enzymes such as peroxidase (DIAS-ARIEIRA et al., 2013), promoting carotenoid degradation (UENOJO et al., 2007). There was a negative correlation (−0.60, P < 0.05) between total carotenoid and protein contents, suggesting that the increase in protein content affected carotenoid accumulation in roots.
The TPC (24.77-27.97 mg GAE g −1 ) and antioxidant activity were not influenced by M. javanica inoculum density (P > 0.10). DPPH activity was 0.01 μmol Trolox g −1 , regardless of inoculum density. According to the FRAP assay, all samples had  an antioxidant activity of 0.12 μmol Trolox g −1 , except for T2, which had an activity of 0.14 μmol Trolox g −1 . Similar results were observed in tomato plants subjected to water and nematode stress (ATKINSON et al., 2011). Plants accumulate phenolic compounds as protection against nematode infection (NACZK & SHAHIDI, 2004). These compounds are toxic to the parasite and limit the penetration of nematodes and other pathogenic microorganisms (TALCOTT & HOWARD, 1999). Although, carrot tubers affected by M. javanica had presented visible damage and reduction of carotenoids, the TPC and antioxidant activity were not affected, indicating that they could still be used in the food industry. This strategy can reduce food waste. In a study by GULL et al. (2015), for instance, carrot pomace was used in pasta production as a partial, highly nutritional substitute for wheat.

CONCLUSION
Although, carrot tuber length was not negatively influenced by inoculum density, tuber weight significantly decreased with inoculum densities greater than 2250 nematodes plant −1 . Antioxidant activity, TPC, moisture content, and ash content were not influenced by inoculum density. The decrease in carotenoid content was associated with an increase in protein levels. Root galls caused visible changes to tuber morphology, which negatively affects the vegetable's commercial value. Nevertheless, affected tubers may find application in the food processing industry, as they still have high nutritional quality.