Impact of cover crops on population density of the root-lesion nematode Pratylenchus penetrans.

doi:10.21203/rs.3.rs-3338686/v1

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Research Article

Impact of cover crops on population density of the root-lesion nematode Pratylenchus penetrans.

https://doi.org/10.21203/rs.3.rs-3338686/v1

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published 22 Dec, 2023

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In Belgium and worldwide, agriculture has been under threat from plant-parasitic nematodes. Root-lesion nematodes (Pratylenchus spp.) cause yield loss in many vegetable, arable and horticultural crops. Increased use of cover crops can affect population build ups. This study evaluated the reproductive potential of Pratylenchus penetrans on different cultivars of cover crops both, in pot tests and field experiments. Twenty three different cultivars were evaluated against P. penetrans at low (10 P. penetrans per 100 cm³ soil) and high (100 P. penetrans per 100 cm³ soil) initial inoculum density in a pot experiment under glasshouse conditions. Based on their reproductive factor (Rf) after 8 weeks, they were classified under five different categories: Non-host, Poor host, Maintenance host, Good host, and Excellent host. At low Pi, fodder radish 'Defender' and 'Doublet' and birds foot trefoil 'Lotar' were poor hosts. At high Pi, birds foot trefoil 'Lotar' and 'Franco', fodder radish 'Doublemax', 'Maximus' and 'Contra', black oat 'Pratex', phacelia 'Natra', Italian ryegrass 'Meroa' and 'Fedra', rye 'Dukato' and 'Matador' reduced the P. penetrans population. Under field conditions, fodder radish 'Doublet', Japanese oat 'Delux', yellow mustard 'Chacha', Phacelia 'Natra', Italian ryegrass 'Fedra' and Tagetes patula reduced the root-lesion nematode population. The infectious process of P. penetrans on some of these cultivars proven to be poor host was screened using Cone tainers™. Penetration, development and multiplication of P. penetrans was assessed. The information opens possibilities for further resistance breeding. Based on our results we can conclude that with proper choice of selected cultivars of cover crops, P. penetrans can be controlled but that further breeding efforts are needed.

Host plant status

reproductive factor

infectious process

plant-parasitic nematodes

Plant-parasitic nematodes are a major constraint to agricultural production worldwide. Decraemer and Hunt (2013) estimated over 4000 species of plant-parasitic nematodes (PPN); causing several serious problems to world food production. Haegeman et al. (2012) demonstrated how PPN have developed different strategies that allow them to successfully infect their hosts and multiply. They can reduce both quantity and quality of crops (Manzanilla-Lopez et al., 2004). Severe crop losses can be caused directly through feeding on the plant or indirectly by creating pathways for attack by other opportunistic pathogens where they feed, grow, and multiply. Consequently, it is difficult to accurately estimate yield losses due to nematodes. In most countries, especially in developing countries, many farmers have little or no knowledge of the existence of PPNs and this likely leads to the significant underestimation of the true figures for damage and crop yield losses. Jones et al. (2013) pointed out that growers often misinterpret nematode problems for other diseases because they are soil-borne pathogens and are microscopic in nature. Yield loss was estimated to be US$80 billion (Coyne et al., 2018; Nicol et al., 2011) to $157 billion worldwide (Singh et al., 2015).

Root-lesion nematodes are the third in rank behind root-knot and cyst nematodes for the greatest economic impacts on crops worldwide (Jones et al., 2013) with more than 89 species identified by morphometric means of which many are widely distributed (Castillo & Vovlas, 2007; Subbotin et al., 2008). They differ from root-knot nematodes via their ability to enter and leave the root tissues (migratory endoparasitic nematode) during their life cycle. They penetrate the root tissues for feeding and reproduction (Esteves et al., 2015; Moens & Perry, 2009). All the stages are vermiform. Juvenile stages (apart from J1) and adult stages are migratory and all stages apart from eggs and J1 are capable of invading host plants. Mature females lay eggs either inside infected roots, deposited singly or in groups or rows, or in adjacent soil (Jones & Fosu‐Nyarko, 2014). Their diverse adaptation has enabled them to colonize different ranges of plant species in different environmental conditions within the tropical and temperate zones (Castillo & Vovlas, 2007). The most important species are P. brachyurus, P. coffeae, P. goodeyi, P. loosi, P. neglectus, P. penetrans, P. pratensis, P. scribneri, P. thornei, P. vulnus and P. zeae. In temperate regions P. penetrans is prevalent (Moens & Perry, 2009). Pratylenchus penetrans has a wide host range of over 350 plant species (Duncan & Moens, 2006). It parasitizes cultivated vegetables, arable crops, fruits, green manure crops and many weeds (Slootweg, 1956) and is difficult to manage with crop rotations. Moreover, the susceptibility and resistance of crops for root-lesion nematodes often varies between cultivars of the same crop (Townshend, 1989). Cover crops which allow P. penetrans multiplication include: white clover (Trifolium repens), oat (Avena sativa), rye (Secale cereale), kura clover (Trifolium ambiguum), radish and oilseed (Bélair et al., 2002; Thies et al., 1995). In contrast, P. penetrans does not multiply on cover crop plants classified as poor host to non-host including forage sorghum (Sorghum bicolor), perennial ryegrass (Lolium perenne), sweet clover (Melilotus alba), pearl millet (Pennisetum glaucum), crown vetch (Coronilla varia) and MNGRN-16alfalfa (Medicago sativa) (Thies et al., 1995). Marigolds (Tagetes patula) are known to actively kill P. penetrans and reduce its population with approximately 90% during a field period of 3 months (Pudasaini et al., 2006). Due to their frost sensitivity marigolds need to be sown before mid-July and this makes them less suitable for most vegetable growers.

Cover crops potentially related to nematode-suppressive qualities vary with geographical location and target nematode species (Viaene et al., 2013). They also play an important role in the maintenance of soil health and crop productivity and are stimulated in the framework of agro-ecological measures to increase biodiversity. Due to the efficiency of fertilizers and pesticides that have been readily available on the market (Viaene et al., 2013) their potentials have not been fully exploited.

Due to the wide host range of P. penetrans, the study of host status of cover crops is important when developing management options. Cultivars of a cover crop species may vary in their suppressive effect to a particular nematode species. Therefore, this study evaluated the reproductive potential of P. penetrans on different cover crop cultivars either in field or in pots in the glasshouse. The infectious process of P. penetrans on so me cover crops proven to be poor hosts was studied to enable further development of breeding programs for the management of P. penetrans.

The experiments took place at Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), in Merelbeke, Belgium, in a temperature controlled glasshouse and growth chamber in pots and Cone tainers^TM (RLC4 type, Stuewe and Sons; Wesemael, 2021) respectively. In total, 23 cultivars from 8 cover crop species were used. Maize and fallow were used as a positive and negative control, respectively. More information regarding the cover crop cultivars is given in Table 1. Two naturally P. penetrans infested fields located in West-Flanders in Belgium were used for the field experiments.

1.2.1 Nematode culture

Pratylenchus penetrans, population originating from Kinrooi (Belgium), was used through the whole pot experiment. It was mass cultured and maintained as a pure culture on carrot discs as described by O'Bannon & Taylor (1968). Infective juveniles (all stages) and adults were extracted from infected carrot discs using Baermann funnel technique as described by Baermann (1917) and Viaene et al. 2021. The purity of the culture was tested molecularly using the method described by Waeyenberge etal. (2009).

1.2.2 Preparation of soil and filling pots and Cone tainers^TM

Sandy loamy soil, sterilised at 100 °C for 16 h, was collected from a Belgian field at Merelbeke. This autoclaved soil was later sieved (250 mesh sieve) and mixed with river sand in the ratio of 1:1. For the first host status experiment, plastic pots of 1.5 l (16.5 cm diam.) were filled with 1500 cm³ sterilised mixed soil and used for cover crops black oat, yellow mustard, phacelia, Italian ryegrass, rye, summer oat and fodder radish which were all kept in glasshouse conditions. Pot sizes of 0.72 l (13 cm diam.) filled with 720 cm³ of sterilised mixed soil were used for the second host status experiment with cultivars of bird´s foot trefoil and kept in a growth chamber. Cone tainers ^TM used for the monitoring of the infectious process were filled with 80 cm³ sterilised mixed soil and kept in the growth chamber before sowing the different cover crop cultivars.

1.2.3 Planting, maintenance and nematode inoculation

Seeds of the cover crops used in this research were provided by different companies (Table 1). The cultivars tested for host plant status were sown singly in different pot sizes (see above) with fallow as control. The number of seeds sown per pot was based on the surface area of the pots and according to agricultural practices. Table 1 demonstrates the relation of seed density with plant/cultivar used for the host plant status experiments in pots. The first experiment took place in the glasshouse with 16 h day at 20-23 °C and 8 h night at 16 °C. The second experiment with only the cultivars of bird’s foot trefoil took place in a growth chamber with 16 h day at 20 °C and 8 h night at 18 °C. The plants were randomly distributed and watered upon requirements to compensate evaporation and plant growth.

The nematode population density of the stock suspension collected from the Baermann’s funnels was determined with the aid of a stereomicroscope at 40× magnification. Based on the density, the volumes needed for the different inoculation densities (Pi) were calculated. A high (100 P. penetrans per 100 cm³) and a low (10 P. penetrans per 100 cm³) Pi was used in both experiments. After seed germination, when plants were with 3 true leaves, each pot was inoculated with the Pi (a mixture of juveniles and adults) in 3 holes of 5 cm deep close to the plant roots with the aid of a micropipette. Five replicates per cultivar for each nematode density was used. The host plant status experiments were terminated at 56 days post inoculation (dpi).

Cultivars showing to be poor host or maintenance host to P. penetrans from the pot experiments were used to evaluate the infectious process in Cone tainers^TM. Maize was used as a control. One nematode density (200 P. penetrans per Cone tainers^TM) with 20 replicates for each cultivar and maize was used. Plants were kept in a growth chamber with 16 h light at 20 °C and 8 h dark at 18°C. Inoculation was done when plants were bearing 3 true leaves in 1 hole of 5 cm deep close to the plant roots with the aid of a micropipette. The infectious process was monitored at 2, 7 and 14 dpi and at 56 dpi nematode reproduction was evaluated.

Table 1: Cover crops examined, seed rate used per ha, 1000 seed weight and seed sources.

Common name	Scientific name	Seed density (kg/ha)	Cultivar	1000 seed weight (g)	Seed source
Bird’s foot trefoil	Lotus corniculatus	10	Bull	1.40	Freudeberger, Germany.
			Barguay	1.20	Barenbrug, Netherlands.
			Lotar	1.25	Oseva Seeds, Czech Republic.
			Franco	1.13	Entecra, Italy.
Phacelia	Phacelia tanacetifolia	10	Angelia	1.8	AVEVE, Belgium.
Phacelia	Phacelia tanacetifolia	10	Natra	1.5	AVEVE, Belgium.
Fodder radish	Raphanus sativus	40	Contra	13.2	Petersen, Germany.
			Dacapo	11.1	Petersen, Germany.
			Defender	11.40	Petersen, Germany.
			Doublet	11.60	Joordens, Netherlands.
			Doublemax	11.20	ILVO, Belgium.
			Maximus	12.40	ILVO, Belgium.
Italian ryegrass	Lolium multiflorum	15	Fedra	4.47	ILVO, Belgium.
Italian ryegrass	Lolium multiflorum	15	Meroa	4.84	ILVO, Belgium.
Black oat	Avena strigosa	40	Delux	15.22	AVEVE, Belgium.
			Amazone	15.32	Limagrain, Belgium.
			Pratex	15.28	AVEVE, Belgium.
Rye	Secale cereale	70	Dukato	36.52	AVEVE, Belgium.
Rye	Secale cereale	70	Matador	36.41	AVEVE, Belgium.
Summer oat	Avena sativa	100	Effectiv	37.37	AVEVE, Belgium.
Summer oat	Avena sativa	100	Simphony	37.23	AVEVE, Belgium.
Yellow mustard	Sinapis alba	20	Chacha	5.3	AVEVE, Belgium.
Yellow mustard	Sinapis alba	20	Carnaval	5.49	Limagrain, Belgium

1.2.4 Nematode extraction and quantification.

1.2.4.1 Pot experiments for host plant status

At the end of the experiments, the population density of P. penetrans was estimated in the organic (roots) and mineral (soil) fraction per pot. Root samples were carefully separated from the soil, washed and weighed to determine fresh weight. Nematodes were later extracted from these roots after they were chopped in small pieces of about 2 cm in length and macerated at high speed in a laboratory blender (Waring commercial) for 60 seconds. These macerated roots were then passed through a sieve of 850 μm into a 1000 ml plastic beaker. After homogenization of the soil sample, a subsample of 200 cm³ of soil was taken and washed through a 850 μm sieve into a 1000 ml plastic beaker. The root and soil samples were separately subjected to automated zonal centrifugation (AZC) (Hendrickx, 1995) to extract the nematodes. The nematode counts of the extract were used to calculate the final population (Pf) and reproductive factor (Rf) per plant/pot.

1.2.4.2 Cone tainers^TM experiment for infectious process

At different time points, viz. 2, 7 and 14 days post inoculation (dpi), the penetration/infectious process was monitored after nematode staining in root tissue with the fuchsin acid method described by Byrd Jr et al. (1983). Maize was used here as a control. At 14 dpi nematodes were also extracted from the soil samples. At 56 dpi nematodes were extracted from the whole root system and soil using the AZC machine as described above.

1.2.5 Field experiment

Two fields, located in West-Flanders in Belgium and naturally infested with P. penetrans, were used to examine the host status of some cover crop cultivars under field conditions. The presence of P. penetrans was confirmed molecularly (Waeyenberge etal., 2009). Based on the soil analysis at a depth of 0-30 cm, soil type in both fields was sandy. The field in Ruddervoorde (N: 51.06226, E: 3.19708) had a pH of 5.2 with a carbon percentage (%C) of 1.1 and in Ruiselede (N: 51.0409, E: 3.3967) the pH was 4.9 with %C of 2.16. The fields were divided into 24 (21 × 20 m) and 18 (3 x 125 m) equal plots respectively. Soil samples were taken per plot to estimate a Pi before any cover crop was sown. This was done with the aid of an auger (25 cm deep and 1.75 cm diam.) by taking 60 soil cores from each plot following a zigzag sampling pattern. Cover crops used in the field experiments were fodder radish 'Doublemax', 'Doublet' and 'Defender', yellow mustard 'Chacha', 'Maryna' and 'Carnaval', Italian ryegrass 'Fedra' and 'Meldela', black oat 'Pratex' and 'Delux', rye 'Dukato', summer oat 'Effectiv', phacelia 'Natra' and 'Angelia' and Tagetes patula 'Labyrinth'. Before the plots were prepared for the next crops, soil samples were taken as describe above to determine the Pf. The cover crop field duration is shown in Table 7. The nematodes were extracted from the collected soil samples with the aid of an AZC as described above and counted with the aid of a binocular microscope.

1.2.6 Data analysis

The data for host status and infectious process for P. penetrans on cover crops were subjected to analysis of variance (ANOVA) using software program SAS. Fisher’s least significant differences (LSD) at P ≤ 0.05 was used to compare the differences among treatments (mean comparison) and data was normalized by log transformation (logx+1).

The formula Rf = Pf/Pi was used to calculate the nematode reproductive factor for each cover crop. Based on the Rf values, the cover crop cultivars were put under five different categories (modified from Ferris et al., 1993) as follows: non-host = (Rf < 0.15), poor host = (Rf < 1.0 ≥ 0.15), maintenance host = (Rf ≤ 2.0 ≥ 1.0), good host (Rf ≤ 4.0 ≥ 2.0) and excellent host (Rf > 4.0).

1.3.1 Host status in pots

The reproduction factor (Rf = Pf/Pi) was used to determine the host status of different cover crops for P. penetrans after inoculation with a low and high initial nematode density. Temperatures in both glasshouse and growing chamber were recorded with a data logger (Testo) to calculate the amount of degree days (DD with 5.1°C as base temperature for P. penetrans) during the period from inoculation to harvest. We registered 937 DD_5.1 in the glasshouse and 1268 DD_5.1 in the growth chamber, which was sufficient for P. penetrans to complete its life cycle (Duncan & Moens, 2006).

The cover crop cultivars showed varying levels of host status observed by reproduction (Tables 2 and 3). At low Pi, fodder radish 'Contra', black oat 'Delux', yellow mustard 'Chacha' and 'Carnaval', Italian ryegrass 'Fedra', rye 'Matador' and summer oat 'Simphony' and 'Effectiv' were all excellent hosts. Fodder radish 'Maximus', phacelia 'Angelia' and 'Natra' and rye 'Dukato' were good hosts. Fodder radish 'Doublemax' and 'Dacapo', black oat 'Amazone' and 'Pratex' and Italian ryegrass 'Meroa' were maintenance hosts. Only fodder radish 'Defender' and 'Doublet' were poor hosts. At high Pi, the host status reduced for most cover crop cultivars except for fodder radish 'Defender' and 'Doublet' which became maintenance host and black oat 'Pratex' which was a good host at high Pi as seen in Table 2. The only cover crops being good hosts at high Pi were black oat 'Pratex' and summer oat 'Simphony'. In the fallow control no nematodes were retrieved at the end of the experiment, eight weeks after inoculation.

When looking at the number of nematodes per gram of root there were no differences between cultivars of the same crop at high Pi (Table 3). At low Pi, significantly more P. penetrans were found per gram of roots of black oat 'Delux' compared to 'Amazone' and 'Pratex'. Also for rye a significant difference between the cultivars was seen. No correlation was observed between nematode reproduction rate and the root weight of cover crops with high nematode numbers being equally recovered from cultivars with low root weight and higher root weight. The highest number of P. penetrans per gram of root was found in phacelia 'Natra' for low Pi and in phacelia 'Angelia' at high Pi.

To enable better comparison between cultivars of the same cover crop, Pf values were also compared between cultivars of the same crop (Fig. 1 and Fig. 2) for both low and high Pi. Phacelia, yellow mustard and summer oat did not show significant differences between cultivars at both, low and high Pi. For fodder radish only significant differences were seen at low Pi where 'Contra' showed a significant higher Pf than 'Doublet'. At low Pi, black oat 'Delux' had a significant higher Pf compared to 'Pratex' and 'Amazone' and rye 'Matador' had a significant higher Pf compared to 'Dukato'. The Pf on Italian ryegrass 'Fedra' was significantly higher than on 'Meroa' at both, low and high Pi.

Table 2: Reproductive factor (Rf ± standard error) of Pratylenchus penetrans Kinrooi population on different cover crops at 56 days post inoculation with initial nematode densities of 10 P. penetrans / 100 cm³ (low Pi) and 100 P. penetrans / 100 cm³ (high Pi). A negative control (fallow) was subjected to the same inoculation. Different letters per column indicate significant differences between cultivars (P ≤ 0.05).

Crop	Cultivar name	Low Pi		High Pi
		Rf	Host status	Rf	Host status
		Rf	Host status	Rf	Host status
Fodder radish	Doublemax	1.30 ± 0.31 b	Maintenance host	1.20 ± 0.12 ab	Maintenance host
	Maximus	2.18 ± 0.56 b	Good host	0.97 ± 0.18 ab	Poor host
	Contra	4.73 ± 1.94 b	Excellent host	0.76 ± 0.13 ab	Poor host
	Dacapo	1.75 ± 0.45 b	Maintenance host	1.29 ± 0.38 ab	Maintenance host
	Defender	0.98 ± 0.40 b	Poor host	1.25 ± 0.15 ab	Maintenance host
	Doublet	0.53 ± 0.22 b	Poor host	1.01 ± 0.14 ab	Maintenance host
Black oat	Delux	144.71 ± 110.90 a	Excellent host	1.98 ±1.32 ab	Maintenance host
	Amazone	1.46 ± 0.50 b	Maintenance host	0.70 ± 0.56 ab	Poor host
	Pratex	1.05 ± 0.35 b	Maintenance host	2.05 ± 1.01 ab	Good host

Table 2(Continued)

Crop	Cultivar name	Low Pi		High Pi
		Rf	Host status	Rf	Host status
		Rf	Host status	Rf	Host status
Yellow mustard	Chacha	6.08 ± 2.59 b	Excellent host	1.06 ± 0.12 ab	Maintenance host
Yellow mustard	Carnaval	14.99 ± 2.72 b	Excellent host	1.25 ± 0.19 ab	Maintenance host
Phacelia	Angelia	3.66 ± 3.15 b	Good host	1.92 ± 0.55 ab	Maintenance host
Phacelia	Natra	3.91 ± 1.38 b	Good host	0.44 ± 0.19 ab	Poor host
Italian ryegrass	Fedra	5.39 ± 2.42 b	Excellent host	0.66 ± 0.28 ab	Poor host
Italian ryegrass	Meroa	1.06 ± 0.39 b	Maintenance host	0.12 ± 0.05 b	Non-host
Rye	Dukato	2.99 ± 1.38 b	Good host	0.44 ± 0.10 ab	Poor host
Rye	Matador	12.06 ± 3.48 b	Excellent host	0.23 ± 0.09 ab	Poor host
Summer oat	Simphony	8.85 ± 6.08 b	Excellent host	2.48 ± 1.86 a	Good host
Summer oat	Effectiv	49.18 ± 32.56 b	Excellent host	1.72 ± 1.05 ab	Maintenance host
Fallow		0.00 ± 0.00		0.00 ± 0.00

Table 3: Final population density of Pratylenchus penetrans per gram of roots (Pf ± standard error), root weight (mean ± standard error) and final nematode population (Pf ± standard error) on different cover crops at 56 days post inoculation with initial nematode densities of 10 P. penetrans / 100 cm³ (low Pi) and 100 P. penetrans / 100 cm³ (high Pi). Different letters per column for root weight (g) and Pf per gram of roots indicate significant differences between cultivars per cover crop (P ≤ 0.05). Different letters per column for final population (Pf) indicate significant differences between all cultivars (P ≤ 0.05)

Crop	Cultivar name	Low Pi			High Pi
Crop	Cultivar name	Root weight (g)	Final population (Pf)	Pf (nematodes / gram of roots)	Root weight (g)	Final population (Pf)	Pf (nematodes / gram of roots)
Fodder radish	Doublemax	14.4 ± 1.38 a	195.4 ± 45.82 a	15.4 ± 5.24	17.1 ± 4.54 ab	1804.6 ± 180.87 ab	181.0 ± 87.20
	Maximus	8.1 ± 1.19 ab	328.2 ± 83.40 ab	45.5 ± 14.61	17.1± 1.39 ab	1457.0 ±266.99 ab	88.8 ± 19.06
	Contra	14.5 ± 3.99 a	708.8 ± 291.35 ab	110.0 ± 83.62	14.1 ± 1.51 ab	1141.6 ± 187.81 ab	87.4 ± 22.26
	Dacapo	5.3 ± 0.46 b	263.0 ± 67.40 ab	53.0 ± 15.84	17.7 ± 2.91 ab	1932.4 ± 550.61 ab	124 1 ± 43.22
	Defender	14.0 ± 2.65 a	146.4 ± 60.29 ab	11.0 ± 5.19	10.2 ± 1.90 b	1883.4 ± 223.19 ab	223.7 ± 63.70
	Doublet	9.1 ± 2.11 ab	79.2 ± 33.37 ab	13.4 ± 7.49	20.9 ± 4.40 a	1513.4 ± 207.77 ab	114.9 ± 55.98
Black oat	Delux	53.4 ± 6.39	4351.0 ± 2157.63 ab	327.3 ± 225.18 a	33.5 ± 4.87 a	2968.2 ± 1985.97 ab	108.9 ± 72.15
	Amazone	110.8 ± 63.40	218.8 ± 71.22 ab	3.2 ± 1.09 b	10.9 ± 0.80 b	1050.2 ± 843.92 ab	121.2 ± 103.75
	Pratex	33.6 ± 1.20	157.6 ± 52.70 ab	4.8 ± 1.65 b	17.9 ± 0.58 b	3078.6 ± 1510.15 ab	181.7 ± 95.84
Yellow mustard	Chacha	3.2 ± 0.69	912.2 ± 387.86 ab	355.5 ± 168.94	5.3 ± 2.36	1592.6 ± 179.28 ab	468.2 ± 94.66
Yellow mustard	Carnaval	5.4 ± 0.79	2249.2 ± 407.75 b	422.9 ± 72.2	9.3 ± 3.24	1878.0 ± 291.58 ab	329.8 ± 110.42

Table 3(Continued)

Crop	Cultivar name	Low Pi			High Pi
Crop	Cultivar name	Root weight (g)	Final population (Pf)	Pf of nematodes per gram of roots	Root weight (g)	Final population (Pf)	Pf of nematodes per gram of roots
Phacelia	Angelia	1.8 ± 1.03	549.4 ± 473.09 ab	175.0 ± 82.09 a	3.2 ± 0.77	2883.2 ± 818.61 b	1871.9 ± 1094.02
Phacelia	Natra	3.0 ± 1.55	587.2 ± 206.99 ab	1028.6 ± 731.56 a	1.0 ± 0.52	656.0 ± 281.63 ab	441.1 ± 236.01
Italian ryegrass	Fedra	14.8 ± 1.50	809.0 ± 363.44 ab	50.9 ± 17.18 a	13.8 ± 4.82	988.0 ± 421.13 ab	202.3 ± 164.75
Italian ryegrass	Meroa	28.5 ± 5.87	158.6 ± 59.03 ab	8.9 ± 5.23 b	13.8 ± 2.03	186.6 ± 69.63 a	16.1 ± 7.46
Rye	Dukato	17.5 ± 7.095	448.2 ± 206.44 ab	28.5 ± 7.5 b	11.5 ± 1.36	656.2 ±151.57 ab	59.3 ± 13.29
Rye	Matador	13.0 ± 2.64	1809.4 ± 522.55 b	141.3 ± 28.64 a	12.9 ± 3.34	353.6 ±134.16 ab	34.1 ± 12.58
Summer oat	Simphony	30.9± 2.68	1326.8 ± 911.52 ab	52.2 ± 38.15 a	30.9 ± 3.71 b	3722.2 ± 2789.05 ab	150.2 ± 91.32
Summer oat	Effectiv	30.6 ± 5.29	7377.2 ± 4884.68 ab	199.9 ± 38.15 a	12.2 ± 1.88 a	2576.6 ± 1579.60 ab	290.2± 212.40
Fallow		0.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00

Data obtained in the second experiment for host plant status (growth chamber), classified bird´s foot trefoil 'Lotar' as poor host while 'Bull', 'Barguay', 'Franco' were maintenance hosts at low initial Pi (Table 4). While at high Pi, 'Franco' and 'Lotar' were poor hosts; 'Bull' and 'Barguay' were maintenance hosts. No correlation was observed between nematode reproduction rate and the root weight of cover crops with high nematode numbers being equally recovered from cultivars with low root weight and higher root weight (Table 5).

Table 4: Reproductive factor (Rf ± standard error) of Pratylenchus penetrans on Bird´s foot trefoil cultivars extracted at 56 dpi with initial nematode density of 10 P. penetrans/100 cm³ (low Pi) and 100 P. penetrans/100 cm³ (high Pi). A negative control (fallow) was subjected to the same inoculation. Different letters per column indicate significant differences between cultivars (P ≤ 0.05).

Crop	Cultivar name	Low Pi		High Pi
Crop	Cultivar name	Rf	Host status	Rf	Host status
Bird’s foot trefoil	Bull	1.56 ± 0.14 a	Maintenance host	1.15 ± 0.17	Maintenance host
	Barguay	1.04 ± 0.14 ab	Maintenance host	1.01 ± 0.40	Maintenance host
	Lotar	0.78 ± 0.18 b	Poor host	0.54 ± 0.06	Poor host
	Franco	1.10 ± 0.32 ab	Maintenance host	0.75 ± 0.15	Poor host
Fallow		0.0 ± 0.01		0.0 ± 0.00

Table 5: Final population density of Pratylenchus penetrans per gram of roots (Pf ± standard error), root weight (mean ± standard error) and final P. penetrans population density (Pf ± Standard error) on bird´s foot trefoil cultivars extracted at 56 dpi with initial nematode density of 10 J2/100 cm³ (low Pi) and 100 J2/100 cm³ (high Pi). Different letters per column indicate significant differences between cultivars(P ≤ 0.05).

Crop	Cultivar name	Low Pi			High Pi
Crop	Cultivar name	Root weight (g)	Final population (Pf)	Pf of nematodes per gram of roots	Root weight (g)	Final population (Pf)	Pf of nematodes per gram of roots
Bird’s foot trefoil	Bull	19.6 ± 0.80 ab	112.2 ± 10.15 a	5.8 ± 0.60	22.4 ± 1.87	825.2 ± 120.63	37.5 ± 5.82
	Barguay	19.9 ± 4.12 ab	75.1 ± 10.08 ab	4.7 ± 1.08	19.5 ± 2.34	724.6 ±285.91	39.2 ± 14.46
	Lotar	16.6 ± 1.52 b	56.2 ± 12.98 b	3.6 ± 0.93	18.9 ± 2.05	385.5 ± 43.57	20.8 ± 2.21
	Franco	25.6 ± 2.37 a	79.4 ± 22.94 ab	3.4 ± 1.16	22.1 ± 2.78	541.9 ± 106.52	26.2 ± 5.49
Fallow			1.2 ± 0.80			2.0 ± 1.26.

1.3.2 Infectious process

Based on the results from the pot experiments for host plant status, black oat 'Amazone', phacelia 'Natra', Italian ryegrass 'Meroa' and 'Fedra', rye 'Dykato' and 'Matador' and bird’s foot trefoil 'Franco' were further screened to assess the penetration of P. penetrans at 2, 7 and 14 dpi. At 14 dpi, nematodes were quantified not only from the root fraction (Fig. 3) but also from the mineral fraction (Fig. 4). At 56 dpi the Pf was determined.

At 2 dpi the significant highest number of P. penetrans was found in the roots of rye 'Dukato' whereas the lowest number was seen in rye 'Matador' followed by black oat 'Amazone' (Fig. 3). Penetration in rye 'Matador' increased and rendered the significantly highest number of P. penetrans in the roots at 7 dpi followed by Italian ryegrass 'Amazone', rye 'Dukato' and the control maize. The lowest number at 7dpi was found in Italian ryegrass 'Meroa' and 'Fedra', phacelia 'Natra' and bird’s foot trefoil 'Franco'. At 14 dpi the highest number of nematodes in the roots was found in bird’s foot trefoil 'Franco' which was significantly different from the rest of the tested cover crop cultivars and control. Italian ryegrass 'Meroa' showed the second highest number of nematodes in the roots followed by phacelia 'Natra' and rye 'Dukato' which were all not significantly different from each other. The lowest penetration was found on the control Maize followed by Italian ryegrass 'Fedra', black oat 'Amazone' and rye 'Matador'.

The number of P. penetrans found in the soil was extracted at 14 dpi and is shown in Fig. 4 combined with the final population (Pf) which was a combination of the nematodes in the roots and the nematodes found in the soil. The highest number of P. penetrans found in soil (mineral fraction) was seen for Italian ryegrass 'Fedra', black oat 'Amazone' and phacelia 'Natra'. The lowest number of P. penetrans found in soil was with rye 'Dukato' and 'Matador', followed by maize and 'Franco'. The highest Pf at 14 dpi (Fig. 4) was found in bird’s foot trefoil 'Franco' which was significantly different from the rest of the cultivars including the control maize. The significantly lowest Pf was found in the maize cultivar.

The final population (Pf) and the reproductive factor (Rf) was examined at 56 days dpi. Multiplication was significantly highest in the susceptible control maize. Rye 'Dukato' and Italian ryegrass 'Fedra' were both maintenance hosts. The lowest multiplication was seen in black oat 'Amazone', bird’s foot trefoil 'Franco', phacelia 'Natra', Italian ryegrass 'Meroa' and rye 'Matador' which all are classified as poor host.

Table 6: The mean final nematode population (Pf ± Standard error) and respective mean reproductive factor (Rf ± Standard error) and host status at 56 dpi with initial nematode density of 200 Pratylenchus penetrans. Different letters per column indicate significant differences between plants (P ≤ 0.05).

Cover crop	Cultivar name	Pf	Rf	Host status
Black oat	Amazone	71.9 ± 8.44 d	0.36 ± 0.04 d	Poor host

Phacelia	Natra	151.6 ± 14.11 cd	0.76 ± 0.07 cd	Poor host

Italian ryegrass	Meroa	182.2 ± 11.48 bcd	0.91 ± 0.06 bcd	Poor host
Italian ryegrass	Fedra	219.1 ± 18.1 bc	1.09 ± 0.09 bc	Maintenance host

Rye	Dukato	283.6 ± 45.65 b	1.42 ± 0.23 b	Maintenance host
Rye	Matador	184.4 ± 17.81 bcd	0.92 ± 0.09 bcd	Poor host

Bird´s foot trefoil	Franco	120.2 ±16.95 cd	0.60 ± 0.08 cd	Poor host

Maize	LG 30.211	648.3 ± 84.47 a	3.24 ± 0.42 a	Good host

1.3.3 Host status screening tests in field

Based on the availabilities of seeds together with the results from the pot trials some cultivars were selected to be tested under field conditions (Table 7). Due to the different growing seasons of some of this cover crop cultivars, there was a considerable variation in growth and germination. Plots having a greater coverage with cover crops exceeding 90% were monitored. Cover crop black oat 'Pratex' was an excellent host based on its Rf value (Table 7) followed by Italian ryegrass 'Meldela' as a good host. Rye 'Dukato', yellow mustard 'Maryna' and phacelia 'Angelia' were maintenance host. A non-host was found in black oat 'Delux' while the rest of the tested cover crop cultivars were poor host.

Table 7: The host status of different cover crops for Pratylenchus penetrans under field conditions based on the reproductive factor (Rf).

Cover crop	Cultivar	Date of sowing	Field growth duration (weeks)	Seed density	Degree days (DD_5.1)	Pi	Rf	Host status	Previous crop sown/grown


Fodder radish	Doublemax	14/08/2019	33	24 kg/ha	1251.5	73.67	174.88	Excellent host	Spinach (Whale)

	Doublet	12/06/2020	37	24 kg/ha	1494.625	340.33	0.41	Poor host	Spinach + Fodder radish (Doublet)

						214	0.55	Poor host	Pea (Geneva) + Black oat (Pratex)

						226.33	0.55	Poor host	Pea (Pixia) + Black oat (Pratex)

						148.67	0.09	Non-host	Carrots (Nerac) + Fallow

						166.67	0.35	Poor host	Leek (Krypton) + Fallow

						111.33	0.74	Poor host	Leek (Poulton) + Fallow

		18/08/2021	37	24 kg/ha	-	333.67	0.29	Poor host	Spinach (Meerkat)

		13/10/2021	22	12 kg/ha	-	50	1.15	Maintenance host	Bean (Zembla)

						40	3.14	Good host	Bean (Zembla)

	Defender	29/04/2020	11	12 kg/ha	1001.604	236.33	0.18	Poor host	Italian ryegrass (Meldela)

						375.67	0.19	Poor host	Italian ryegrass (Meldela)

						90.33	0.74	Poor host	Italian ryegrass (Meldela)

Table 7 (Continued)

Cover crop	Cultivar	Date of sowing	Field growth duration (weeks)	Seed density	Degree days (DD_5.1)	Pi	Rf	Host status	Previous crop sown/grown


Italian ryegrass	Fedra	12/06/2020	37	50 kg/ha	1494.625	340.33	0.26	Poor host	Spinach (Meerkat) + Fodder radish (Doublet)

						214	0.07	Poor host	Pea (Geneva) + Black oat

						226.33	0.08	Non-host	Pea (Pixia) + Black oat

						148.67	0.03	Non-host	Carrot (Nerac) + Fallow

						166.67	0.19	Poor host	Leek (Krypton) + Fallow

						111.33	0.44	Poor host	Leek (Poulton) + Fallow

		18/08/2021	-	50 kg/ha	-	184	0.24	Poor host	Spinach (Meerkat)

	Meldela	26/11/2019	16	30 kg/ha	535.57	71	2.06	Good host	Leek (Krypton)

						8.33	4.39	Excellent host	Leek (Poulton)

Rye	Dukato	26/11/2019	16	100kg/ha	535.57	56.67	0.69	Poor host	Leek (Krypton)

						53	1.08	Maintenance host	Leek (Poulton)

Summer oat	Effectiv	29/04/2020	11	100 kg/ha	1001.604	115.67	0.42	Poor host	Black oat (Pratex)

						304	0.62	Poor host	Black oat (Pratex)

						71.67	1.5	Maintenance host	Black oat (Pratex)

Table 7 (Continued)

Cover crop	Cultivar	Date of sowing	Field growth duration (weeks)	Seed density	Degree days (DD_5.1)	Pi	Rf	Host status	Previous crop sown/grown


Phacelia	Natra	12/06/2020	37	16 kg/ha	1494.625	340.33	0.78	Poor host	Spinach (Whale) + Fodder radish (Doublet)

						214	0.003	Non-host	Pea (Geneva) + Black oat (Pratex)

						226.33	0.005	Non-host	Pea (Pixia) + Black oat (Pratex)

						148.67	0.31	Poor host	Carrot (Nerac) + Fallow

						166.67	1.42	Maintenance host	Leek (Krypton) + Fallow

						111.33	3.34	Good host	Leek (Poulton) + Fallow

		13/10/2021	22	8 kg/ha	-	75	1.42	Maintenance host	Leek (Krypton) + Fallow

						43	3.34	Good host	Leek (Poulton) + Fallow

						31.33	0.91	Poor host	Leek (Krypton)

						86.67	0.67	Poor host	Leek (Krypton)

	Angelia	29/04/2020	45	8 kg/ha	2574.718	184.33	0.56	Poor host	Rye (Dukato)

						258.67	0.57	Poor host	Rye (Dukato)

						77.67	2.04	Good host	Rye (Dukato)

		30/09/2020	23	8 kg/ha	513.34	38.33	2.37	Good host	Pea (Pixia)

Table 7 (Continued)

Cover crop	Cultivar	Date of sowing	Field growth duration (weeks)	Seed density	Degree days (DD_5.1)	Pi	Rf	Host status	Previous crop sown/grown


Yellow mustard	Chacha	12/06/2020	37	30 kg/ha	1494.625	340.33	0.44	Poor host	Spinach (Whale) + Fodder radish (Doublet)

						214	0.31	Poor host	Pea (Geneva) + Black oat (Pratex)

						226.33	0.41	Poor host	Pea (Pixia) + Black oat (Pratex)

						148.67	0.36	Poor host	Carrot (Nerac) + Fallow

						166.67	0.16	Poor host	Leek (Krypton) + Fallow

						111.33	0.33	Poor host	Leek (Poulton) + Fallow

		18/08/2021	-	20 kg/ha	-	197.67	0.66	Poor host	Spinach (Meerkat)

	Maryna	30/09/2020	23	10 kg/ha	513.34	34.67	1.87	Maintenance host	Summer fallow

						44.33	1.048	Maintenance host	Summer fallow

						29.33	1.195	Maintenance host	Summer fallow

	Carnaval	30/09/2020	23	10 kg/ha	513.34	46.33	5.075	Excellent host	Pea (Pixia)

Table 7 (Continued)

Cover crop	Cultivar	Date of sowing	Field growth duration (weeks)	Seed density	Degree days (DD_5.1)	Pi	Rf	Host status	Previous crop sown/grown


Tagetes patula	Labyrinth	12/06/2020	37	30 kg/ha	1494.625	340.33	0.104	Non-host	Spinach (Whale) + Fodder radish (Doublet)

						214	0.06	Non-host	Pea (Geneva) + black oat (Pratex)

						226.33	0.07	Non-host	Pea (Pixia) + black oat (Pratex)

						148.67	0.007	Non-host	Carrot (Nerac) + Fallow

						166.67	0.004	Non-host	Leek (Krypton) + Fallow

						111.33	0.005	Non-host	Leek (Poulton) + Fallow

		9/07/2021	36	8 kg/ha	-	136.67	0.04	Non-host	Spinach (Eland)

						123.67	0.035	Non-host	Spinach (Eland)

						84.33	0.6	Non-host	Spinach (Eland)

						55.33	0.11	Non-host	Spinach (Eland)

		18/08/2020	29	8 kg/ha	-	33.67	2.97	Good host	Summer oat (Effectiv)

						61.67	2.51	Good host	Summer oat (Effectiv)

						99	1.11	Maintenance host	Summer oat (Effectiv)

Table 7 (Continued)

Cover crop	Cultivar	Date of sowing	Field growth duration (weeks)	Seed density	Degree days (DD₅.₁)	Pi	Rf	Host status	Previous crop sown/grown


Black oat	Pratex	14/08/2019	33	80 kg/ha	1206.42	52.33	5.77	Excellent host	Pea (Geneva)

						215	1.87	Maintenance host	Pea (Pixia)

		18/08/2021	-	80 kg/ha	-	71.67	0.31	Poor host	Pea (Geneva)

		13/10/2021	22	80 kg/ha	-	53.33	0.86	Poor host	Bean (Zembla)

	Delux	12/06/2020	37	80 kg/ha	1494.625	340.33	0.25	Poor host	Spinach (Meerkat) + Fodder radish (Doublet)

						214	0.07	Non-host	Pea (Geneva) + Black oat (Pratex)

						226.33	0.07	Non-host	Pea (Pixia) + Black oat (Pratex)

						148.67	0.05	Non-host	Carrot (Nerac) + Fallow

						166.67	0.011	Non-host	Leek (Krypton) + Fallow

						111.33	0.0235	Non-host	Leek (Poulton) + Fallow

		18/08/2021	-	80 kg/ha	-	99.67	0.61	Poor host	Pea (Geneva)

		13/10/2021	22	80 kg/ha	-	50.67	0.36	Poor host	Bean (Zembla)

						50.67	0	Non-host	Bean (Zembla)

The impact of cover crop cultivars on different density levels of P. penetrans was determined by comparing the nematode Rf. The result of this study showed that the performance of different cover crops against P. penetrans is different depending on cultivar and initial population density. For yellow mustard, phacelia, Italian ryegrass, rye and summer oat, the host plant status at high Pi was lower than at low Pi. This shows that initial Pi plays an important role in whether a P. penetrans population will increase or decrease on a certain cover crop cultivar. The carrying capacity effect at higher initial inoculum densities limits nematode multiplication through resource and space competition. It is known that at high population densities nematode multiplication is limited by competition and the total amount of food that the host can supply (Schomaker & Been, 2013). However, for two cultivars of fodder radish and one black oat cultivar we observed an increase in final population at higher Pi in the pot test. This could not be explained by a better developed root system as there was no difference with the other cultivars. For fodder radish 'Doublet' the same observation was made for M. chitwoodi (Taning et al., 2023). In the field experiments the host status of 'Doublet' also ranged from non-host to good host. Fodder radish 'Doublet', as well as the other tested cultivars, has resistance against M. chitwoodi. It could be that this resistance also affects P. penetrans but that it is less stable in 'Doublet' compared to the other cultivars. Grabau et al. (2017) reported that P. penetrans and Tylenchorhynchus spp. population densities were increased by fodder radish 'Defender' compared to other cover crops or fallow tested in field conditions. In our field trials 'Defender’ was rather reducing the population of P. penetrans but in the pot test it was a poor host at low Pi and a maintenance host at high Pi based on the Rf values. It is clear from our pot test and field trials that a difference in Pi can render different results. The cover crop performance can also change due to environmental and climatological impact, nematode populations present in the field, population density and a combination of factors. Due to the wide host range, it is difficult to manage P. penetrans on crops like rye, rape, oats and perennial ryegrass (Bélair et al, 2002). This was confirmed with the results from our pot trials with two rye cultivars acting as an excellent and good host at low Pi. Oat and rye were reported as excellent hosts for Pratylenchus spp. but the susceptibility depended on cultivar (Townshend, 1989) and initial population density (Grabau et al., 2017). Black oat 'Pratex', which is marketed for the reduction of P. penetrans, showed to be a good host in the pot test and under field conditions. Possibly, the Belgian population of P. penetrans used in the experiments was virulent. Virulence in root-lesion nematodes such as in Pratylenchus coffeae populations coming from different locations in Japan on sweet potato, has been reported by Mizukubo and Sano (1997). Bird’s foot trefoil cultivars were poor hosts to maintenance host. Given their positive effect on M. chitwoodi (Taning et al., 2023) further testing under field conditions is recommended. Townshend & Potter (1976) classified bird’s-foot trefoil as a good host for P. penetrans but a poor host for P. neglectus. From the screening of the infectious process we could see that the number of P. penetrans inside the roots of bird’s foot trefoil 'Franco' at 14 dpi was higher compared to the other tested cover crop cultivars. However, from the pot experiment we found a rather low amount of P. penetrans per gram of root. This could indicate that bird’s foot trefoil can act as a trap crop and should be further examined. The highest number of P. penetrans per gram of root was found in phacelia and to a lesser extend in yellow mustard. High penetration of phacelia 'Natra' was seen in the screening of the infectious process. The evaluation of the Pf at 56 dpi mostly confirmed the results obtained from the pot test.

Different cover crops from the pot test showing to be a poor host for P. penetrans were also incorporated in a crop rotation scheme under field conditions. In general, most crops gave better results in the field to reduce the P. penetrans population compared to the pot tests. In the field several biotic and abiotic factors will influence the results. It is known that cover crops could produce phytochemicals that control PPNs. Brassicaceae species contain glucosinolates and, when tissues are disrupted, form isothiocyanates that are nematicidal (Chitwood, 2002; Oka, 2010). Ngala et al. (2015) showed that during growth of Brassica juncea and Eruca sativa, viability of Globodera pallida encysted eggs reduced. Tagetes produce polythienyls that suppress nematode populations (Reynolds et al., 2000; Chitwood, 2002; Hooks et al., 2010). Apart from variability in population densities within the fields also the growing period showed to have an effect. Tagetes patula 'Labyrinth' was a non-host when sown in June or beginning of July but showed to be a good host when sown in August. Tagetes patula is known to actively reduce the population of P. penetrans and is also not a host for root-knot nematodes. Evenhuis et al. (2004) showed in a field experiment a significant reduction of the P. penetrans soil population density after growing Tagetes patula. To have optimal results the crop should be on the field for at least three months and as it is frost sensitive it must be sown latest end of July under Belgian climatic conditions. Most likely, the sowing mid-August didn’t allow the marigolds to properly control P. penetrans. In our pot test zero or very low amounts of P. penetrans were retrieved from the fallow control. Up to 24 months of survival of P. penetrans in soil has been reported but this was depending on soil type (Townshend, 1984). Our soil was heat sterilized and this might have influenced survival of the original inoculum.

It is not often easy to control root-lesion nematodes due to their wide host range and their migratory nature. Choosing the right cover crop to incorporate in a rotation will not only reduce the nematode population, but will also increase biodiversity, soil fertility, prevent leaching and improve water holding capacity. In this study, different cover crop cultivars showed potential to be used in rotations. Fodder radish is a host plant for P. penetrans and maintains or (slightly) increases the population. In cultivars with M. chitwoodi resistance, we sometimes also see a decrease in the population of P. penetrans. Therefore, M. chitwoodi resistant cultivars of fodder radish are a good choice if M. chitwoodi is also present in the field. Bird’s foot trefoil is a moderate host plant for P. penetrans and will maintain or slightly decrease the population when the initial population is high. However, the cultivation is very sensitive to weeds and weed control is necessary. Phacelia is a host plant for P. penetrans and increases numbers when initially low numbers of P. penetrans are present. At higher initial populations, the population remained stable or a decrease in the numbers of P. penetrans was observed. Yellow mustard is a host for P. penetrans and will increase the population when a low initial population is present. With higher numbers of P. penetrans the population is maintained or may decrease slightly. The effect of black oats on the population of P. penetrans is cultivar dependent. In certain cultivars the population decreases while it increases in other cultivars, especially with low numbers of P. penetrans. Italian ryegrass is a host plant for P. penetrans and maintains or increases the population when initially low numbers of P. penetrans are present. A decrease in the numbers of P. penetrans was observed in higher initial populations. Rye is a moderate host plant for P. penetrans and will increase the population at low numbers. Marigold (Tagetes patula cultivars) are not a host plant for P. penetrans and also actively kill P. penetrans. To have an optimal effect this crop should be in the field for 3 months and sown at the latest end of July/beginning of August because of frost sensitivity. Good weed control is necessary. Differences between cultivars of cover crops were observed and the screening test of the infectious process showed differences in penetration. This shows that there are options for P. penetrans resistance breeding in cover crops.

Acknowledgement

This study was funded by Flanders Innovation & Entrepreneurship (vlaio, contract HBC.2016.0770).

Ethics declarations

Competing interests

The authors have no competing interests to declare that are relevant to the content of this article.

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