Meloidogyne minor, first reported on potatoes in the Netherlands in 2004, is an emerging nematode pest in Europe. It damages turfgrass, particularly creeping bentgrass (Agrostis stolonifera) grown on sandy soils such as those of golf greens. However, little is known of the nematode's life history and pathology. In this study, the spatial and temporal distribution of M. minor on a creeping bentgrass green in Ireland was determined over a 15 month period. Cores were taken on transects across yellowing patches of grass caused by nematode damage. Second-stage juveniles (J2) were absent from the soil from November to February, when soil temperatures were below 10°C. Both galls and egg masses were present throughout the year but were more abundant in late summer and early autumn. More J2, galls and egg masses were present in the top 10 cm of soil than at a depth of 11–20 cm. The nematode population tended to decrease as distance from the centre of the yellow patches increased. The diameter of visual symptoms (yellow patches) was also recorded over the 15 months. The mean diameter of five sampled patches increased from 23·7 cm in June 2003 to 45·2 cm in August 2004. There were 158–193 galls per 100 cm3 soil at the margin of the visible infested area, indicating that this could be the threshold level for visible symptoms.
Root-knot nematodes, Meloidogyne spp., are amongst the most damaging of plant parasitic nematodes. They can cause extensive root damage, affecting water and nutrient uptake in plants (Karssen & Moens, 2006). With reductions in the use of chemical nematicides, the importance of the genus in Europe is increasing (Wesemael et al., 2011). Meloidogyne minor, first described in 2004 (Karssen et al., 2004), is considered an emerging pest species in Europe (Moens et al., 2009). To date, M. minor has been primarily a pest of creeping bentgrass (Agrostis stolonifera stolonifera) on golf greens and other turf grass amenities in England, Wales and Ireland, where it causes yellow patches (Karssen et al., 2004; Lammers et al., 2006). Reduction of root density due to nematodes can produce depressions in the turf (Fleming et al., 2006; Turner & Fleming, 2006). The resulting unevenness of the turf, together with unsightly yellow patches, reduces the amenity value of the affected courses. Therefore, the disease is a major concern to the golf industry (Fleming et al., 2008). Symptoms are most pronounced on golf greens with a high sand content, including those constructed according to United States Golf Association (USGA) guidelines where the root zone has up to 85% sand (minimum 60%; USGA, 2004). Meloidogyne minor is also a potential threat to certain crop plants. In 2000, the species was found heavily infesting potato roots in a field in the Netherlands (Karssen et al., 2004) and controlled experiments indicate that it can reproduce on tomato, carrot, wheat, barley and oats (Karssen et al., 2004; Lammers et al., 2006). However, in field trials, only potato supported a significant reproduction of M. minor (Thoden et al., 2012). Meloidogyne minor is now known to occur in Britain, Ireland, the Netherlands and Belgium (Viaene et al., 2007; Vandenbossche et al., 2011); however, the full extent of its geographic distribution is unknown (Lammers et al., 2006).
Little is known of the biology of M. minor, and there are no studies on its population dynamics or spatial distribution. Factors such as temperature, soil type, crop management strategies and root spatial distribution can affect nematode population densities and distribution in soil (Franklin et al., 1971; Carpenter & Lewis, 1991; Windham & Barker, 1993; Zhang & Schmitt, 1995). Meloidogyne spp. are sedentary endoparasitic nematodes and deposit all their eggs in egg masses. This results in the nematodes typically exhibiting a highly aggregated and uneven distribution (Goodell & Ferris, 1980). Knowledge of the spatial distribution of nematodes is important in understanding their epidemiology. Population densities are usually enumerated in a composite sample composed of many subsamples (Duncan & Phillips, 2009), providing limited information on the nature of the spatial distribution. Spatial distributions of nematodes have been described in relation to certain discrete host plants (e.g. Zhang & Schmitt, 1995), but studies of nematode spatial distribution in turf grass or pastures are rare. This study determines the spatial and temporal dynamics of M. minor on a USGA golf green over a 15 month period and describes the relationship between the nematodes and visible disease symptoms (yellow patches).
Materials and methods
Sampling was conducted on a golf course nursery in County Kildare in the east of Ireland. The nursery was planted in 2002 with creeping bentgrass (cv. Providence). Five patches of root-knot nematodes were located based on visible symptoms (yellowing) and the presence of galls on roots. The nematodes were identified as M. minor based on morphological criteria and esterase patterns (Karssen et al., 2004). The centre of each patch was permanently marked by a wooden dowel, the top of which was flush with the soil surface. Each patch was sampled monthly from June 2003 to August 2004 (except July 2004). Cores (1·9 cm diameter) were taken to a depth of 20 cm, divided into 0–10 and 11–20 cm portions (total volume of 20 cm core = 45·4 cm3). Cores were taken at 5 cm intervals along a radial transect starting either 5 or 10 cm from the centre of the patch and extending beyond the area with visible symptoms. Each radius was sampled only once. Fewer samples were taken at 5 cm from the centre due to the limited area available at that circumference. The sampled radius was extended over time from 25 to 40 cm, as the patch increased in size. Second-stage juveniles (J2) were extracted using the tray method (Whitehead & Hemming, 1965) for 24 h at 20°C. Following extraction of J2, roots were gently cleaned of adhering soil and blotted dry with tissue paper, the upper 2 cm of root thatch was removed and the remaining roots were weighed. The roots were examined with the aid of a dissecting microscope (×25) and the numbers of galls and egg masses were recorded. When they could be distinguished, the diameters of visible symptoms (yellow patches) were measured at each sampling time; symptoms were most apparent during the summer months. Meteorological data (soil temperature at 10 and 20 cm) were obtained from the Met Éireann Casement climate station, 15 miles (24·1 km) from the field site. Rainfall levels are not presented as golf greens are liberally irrigated.
The effects of soil depth, distance from patch centre and sampling time on number of J2, number of galls, number of egg masses and the percentage of galls with egg masses were analysed using anova on transformed data. The numbers of J2, galls or egg masses were transformed using a square root (x +0·5) transformation. The percentage of galls with egg masses was transformed using an arcsine [square root (n)] transformation. The full data sets were unbalanced, as not all sampling distances were included at each time point; therefore a restricted data set was used in the anova. This balanced data set included data for 10, 15, 20 and 25 cm from the centre of the patch. The full data sets were analysed using general linear models and confirmed the main effects. Residuals were plotted after analyses and found to be normal and homogeneous.
Meloidogyne minor showed distinct temporal and spatial patterns of abundance (Fig. 1). Time of sampling, distance from centre of patch and soil depth all had a highly significant effect on the numbers of J2, galls and egg masses; there were also significant two-way interactions (Tables 1 and 2).
Table 1. Analysis of variance of the effect of distance from the centre of the Meloidogyne minor patch, sampling time and depth on numbers of J2 (10–25 cm from centre of the patch)
Distance (from patch centre)
Depth × Distance
Depth × Time
Distance × Time
Depth × Distance × Time
Table 2. Analysis of variance of the effect of distance from the centre of the Meloidogyne minor patch, time and depth, on numbers of galls per core, numbers of egg masses per core, and percentage of galls with egg masses (data from 10 to 25 cm along radius of the patch)
Percentage of galls with egg masses
Distance (from patch centre)
Depth × Distance
Depth × Time
Distance × Time
Depth × Distance × Time
Seasonal trends in numbers of J2
J2 were absent during the winter months of November 2003 to February 2004, but were detected at all other times, with up to 374 J2 per 100 cm3 soil (Figs 1 and 2c). The seasonal pattern of J2 numbers differed between the upper and lower cores (depth × time, P <0·001; Table 1). For example, 10 cm from the centre of the patch, the number of J2 in the upper core increased steeply in March 2004 and remained high to the end of the sampling period in August of that year, while in the lower core (11–20 cm), the number rose more gradually, and reached a more distinct peak in May 2004 (Fig. 2c). Soil temperature remained below 10°C (the critical temperature for hatching of M. minor; Morris et al., 2011) from October to February; mean daytime temperature first approached 10°C in March at 10 cm depth (Fig. 2a) and in April at 20 cm depth (Fig. 2b). The general trend, shown for all locations across the transect, was for J2 numbers to decline in autumn (September–October 2003) from a peak in August 2003. Then, following the winter absence, numbers increased from March 2004 onwards, reaching higher levels in summer 2004 than in the previous summer at the same location (Figs 1 and 2c).
Seasonal trends in numbers of galls and egg masses
Galls and egg masses were present throughout the year (Fig. 1). Numbers of galls tended to be low in winter and to increase towards late summer, but the exact nature of the change over time depended both on depth in soil and on distance from the centre of the patch (significant interactions, P <0·001; Table 2). At 10 cm from the patch centre, the number of galls in the upper core doubled from June to September 2003, then declined steeply to January, remained comparatively constant until April and rose steeply again to May 2004 (Fig. 2d). Seasonal changes were less pronounced in the lower core (Fig. 2d). The pattern illustrated in Figure 2d is typical for locations towards the centre of the patch (5–20 cm), with two fairly equal peaks around September–October 2003 and June–August 2004, but towards the edge of the patch (25–40 cm) the earlier peak was smaller or absent as the infestation expanded outwards over time (Fig. 1).
There were similar seasonal trends in the abundance of egg masses, but the number of egg masses was notably low in December 2003 (Figs 1b and 2e). The proportion of galls with attached egg masses also varied seasonally (Table 2). There were two peaks, one in October–November 2003 and one in March–April 2004 (Fig. 2f). The November peak of 60% galls with egg masses was followed by a decline to 10% in December 2003. At other times the proportion of galls with egg masses was relatively constant (30–40%).
Spatial distribution of J2
J2 density was strongly influenced both by soil depth and by distance from the centre of the patch (P <0·001, Table 1). There were more J2 in the upper core than the lower core at each sampling date and location. There was a significant depth × time interaction (P <0·001). Usually, there were 2–4 times as many J2 in the upper core as in the lower core, but in March 2004 the difference was tenfold (Fig. 2c).
The number of J2 per core tended to decline towards the edge of the patch (Fig. 1). The exact pattern varied over time (distance × time, P <0·03; Table 1). A steady decline from centre to edge was evident in some months (e.g. August 2004; Fig. 3a), while in others the highest number of J2 occurred at 10, 15 cm or 20 cm from the patch centre, and then declined towards the patch margin. For example, in August 2003, J2 were most abundant at 10 cm from the patch centre (Fig. 3a). The area within which J2 were recovered increased in radius over the sampling period from 15 cm in June 2003 to a maximum of 40 cm in June 2004 (Fig. 1).
Spatial distribution of galls and egg masses
As for J2, numbers of galls and egg masses were high in the centre of the patch and declined towards the edge (Fig. 1). Numbers were also higher in the upper than in the lower core throughout the patch (data not shown). There was a significant depth × distance interaction for both galls and egg masses (P <0·001; Table 2): the decline in numbers from the centre of the patch was steeper in the upper than in the lower core (Fig. 4a,b). The proportion of galls with attached egg masses was influenced by distance but not by depth (Table 2); from 5 to 20 cm from the patch centre, at least 40% of galls had attached egg masses, but this declined to <5% at 35 cm (Fig. 4c), where new galls were forming. Figure 3 shows the spread of the infestation over a 1-year period. In August 2003, galls and egg masses were recorded at a maximum of 25 cm from the centre of the patch. By August 2004 this had increased to 35 cm (Fig. 3b,c).
Relationship between Meloidogyne and visible symptoms
Visible symptoms (primarily yellowing of the grass) could only be seen in the warmer months of the year, when the grass was actively growing. At these times, the diameter of the yellow patch was measured. The patch nearly doubled in size over the 15-month study period, from an average diameter of 23·7 cm in June 2003 to 45·2 cm August 2004 (Table 3). The gall density at the visible patch margin (estimated by linear interpolation from values shown in Table 3) was 159 to 194 galls per 100 cm3 soil in most months, but was lower in August of both years (108 and 150 galls per 100 cm3 soil in 2003 and 2004, respectively). Throughout this study there was no evidence for a recovery in turf quality.
Table 3. Extent of visual symptoms caused by Meloidogyne minor over 15 months, and number of galls per 45 cm3 core at the perimeter of the symptom patch (estimated by linear interpolation between adjacent sampling points)
Diameter of visible symptoms (cm, mean ± SE)
Estimated number of galls at perimeter of visible symptoms
23·7 ± 0·51
27·9 ± 1·43
34·2 ± 1·21
35·9 ± 1·49
32·0 ± 2·23
41·3 ± 1·62
45·2 ± 1·63
Plants infested with Meloidogyne show stunted growth, wilting, and discoloured foliage (chlorosis; Karssen & Moens, 2006). The above-ground symptoms are similar to those caused by a variety of pathogens and nutritional deficiencies; in the case of Meloidogyne, these are explained by the nematodes affecting water and nutrient uptake and translocation by the root system, and interfering with the rate of photosynthesis in leaves (Karssen & Moens, 2006). Root systems may show deformities, most notably the characteristic galls caused by the endoparasitic females.
Meloidogyne minor may result in extensive turf loss when combined with other severe stresses (Fleming et al., 2006), but even the visual symptoms and reduced quality of infested patches have a significant effect on high value turfgrass. When referring to the visible symptoms caused by M. minor, Karssen et al. (2004) stated that ‘on affected greens the patches appeared in new positions each season’. While it is true that new patches may appear, this study shows that symptom patches that are visible in one season reappear in the same location next season, and that the patch of nematodes remains throughout the season. Over a year, visible patches can nearly double in size. Between 159 and 194 galls per 100 cm3 soil were located at the margin of the visible symptoms in most months, indicating that this could be the usual threshold level for these symptoms. There were fewer galls at the patch margin in August of each year, suggesting a lower damage threshold at this time of year. This may be attributed to a combination of increased numbers of egg masses and active egg laying putting an increased demand on the plant (Karssen & Moens, 2006). The visible symptoms were less distinct during the winter months when the M. minor population is lower.
While M. minor is the main root-knot nematode causing problems on golf greens, M. naasi has been found together with M. minor on golf greens (Karssen et al., 2004; Lammers et al., 2006; Viaene et al., 2007). Detailed morphological examination of J2 from 10 affected patches (including the five patches used in the present study) on the golf course where this study took place confirmed that M. minor was present in all of them, and that M. naasi was present as an additional minor component in one. In routine checks on samples of recovered J2 throughout this study, all examined specimens conformed to the description by Karssen et al. (2004). Esterase patterns on a sample of females detected M. minor but not M. naasi (Morris, 2008). While the possibility that some of the Meloidogyne in the study was M. naasi cannot be excluded, the above evidence indicates that the vast majority was M. minor.
This is the first study to document the spatial and temporal distribution of M. minor. Both galls and egg masses were present on bentgrass roots throughout the year. These tended to be more abundant in late summer and early autumn (up to 502 galls per 100 cm3 soil). Following a peak in September 2003, numbers declined steadily until January 2004, presumably due to the death of the previous year's generation. The gradual increase in the number of galls from February to April and the more dramatic increase in the proportion of galls bearing egg masses in March could be due to the nematodes that had entered roots in the autumn maturing and producing egg masses. The larger galls seen in May could have resulted from the invasion of newly hatched J2 from March onwards. There may thus be two generations of M. minor females co-existing: one that resulted in the appearance of J2 from March, and another that developed from those J2. Meloidogyne species typically have many generations, though some species such as M. naasi may have only one (Karssen & Moens, 2006). Factors including food availability and temperature affect the number of generations per year. For example, in England M. naasi completed only one generation per year on wheat, but it was able to complete a second on perennial ryegrass (Franklin et al., 1971). Turfgrass, managed as a perennial crop, provides a constant source of food for nematodes (Crow, 2005). The development of M. minor from egg mass to egg mass can take place in 12 weeks at 15–20°C (Fleming et al., 2006), which would allow for a complete generation to develop during the summer months.
Meloidogyne minor J2 were absent from soil between November and February, despite the constant availability of egg masses from which J2 hatched when incubated at 20°C (Morris et al., 2011). A large number of hatched J2 was present in March, when average daytime temperatures at a depth of 10 cm approached 10°C. As this is the average, temperature at this depth would have exceeded 10°C some of the time. Moreover, temperatures in the upper few centimetres of soil are normally higher, and many of the egg masses were located there. These field data correspond to the results of constant temperature experiments that indicate that the minimum temperature for M. minor hatch lies between 10 and 15°C (Morris et al., 2011). The majority of M. minor was found at a depth of 0–10 cm in the soil, rather than 11–20 cm. The vertical distribution of plant parasitic nematodes is influenced by root distribution (Barker & Imbriani, 1984). The tendency for more nematodes to occur in the upper core may be explained by the greater mass of roots present at this depth compared to 11–20 cm. Usually, there were 2–4 times as many J2 in the upper core, but in March 2004 the difference was tenfold. In March, mean daytime temperatures had reached 10°C at a soil depth of 10 cm but not at 20 cm.
While not specifically designed to aid in prediction or control of the pest, the findings here lead to some preliminary recommendations. The timing of application is important for control measures that target J2, such as mustard-based soil amendments (Fleming et al., 2008). The onset of J2 hatch in spring can be predicted by monitoring soil temperature; when soil temperatures reach 10°C, control agents could be applied. It was found that J2 numbers in the soil remained high for most of the year, without the summer decrease seen in some studies and associated with massive root invasions (Franklin et al., 1971; Belair, 1998). This suggests that reproduction and root invasion rates are in balance for much of the year. As an alternative to the expense of repeated widespread application, the discrete and visible nature of root-knot induced damage lends itself to targeted application. Although symptoms are not obvious throughout the year, it should be possible to adapt methods from precision agriculture (Wrather et al., 2002; Starr et al., 2007) to locate patches in the summer and target application to these areas during the following spring.
The work was funded by a Teagasc Walsh Fellowship. The authors thank Carton House Golf Course for their assistance.