Plant Soil https://doi.org/10.1007/s11104-017-3475-7
REGULAR ARTICLE
Effects of Solanum sisymbriifolium on potato cyst nematode populations in Portugal Margarida C. Dias & Laura S. Perpétuo & Ana T. Cabral & Rosa Guilherme & Maria J. M. da Cunha & Filipe Melo & Óscar C. Machado & Isabel Luci Conceição
Received: 18 April 2017 / Accepted: 25 October 2017 # Springer International Publishing AG 2017
Abstract Background and aims This study was undertaken to evaluate the effects of Solanum sisymbriifolium cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001 on potato cyst nematode (PCN), Globodera spp., populations in the field in Portugal, to improve crop protection systems based on trap crops. Methods Two PCN infested fields (Bolho and Barcouço) were selected. Solanum sisymbriifolium root exudates effects on PCN hatching were evaluated by in vitro assays and their susceptibility/resistance to PCN was assessed. Field trials were performed during 2 years using the following rotation: S. tuberosum cv. Désirée followed by S. sisymbriifolium. Results Solanum sisymbriifolium root exudates promoted hatching of second-stage juveniles (J2). The cultivars tested were resistant to both species of PCN. In both fields, all cultivars had the same behaviour with no statistically significant differences.
Responsible Editor: Duncan D. Cameron. M. C. Dias : L. S. Perpétuo : A. T. Cabral : I. L. Conceição (*) Department of Life Sciences, Centre for Functional Ecology (CFE), University of Coimbra, P-3000 456 Coimbra, Portugal e-mail:
[email protected] R. Guilherme : M. J. M. da Cunha : F. Melo : Ó. C. Machado Department of Agronomical Sciences, School of Agriculture of Coimbra, Research Centre for Natural Resources, Environment and Society (CERNAS), P-3045 601 Coimbra, Portugal
Conclusions As S. sisymbriifolium cultivars were resistant to G. rostochiensis and G. pallida populations in Portugal, they can be included in crop rotations and are potential candidates to incorporate in management programs against PCN. Although there were no statistical differences it seems that fallow could be the best strategy to reduce PCN populations faster and this avoids the introduction of an exotic plant that could become a problem. Keywords Globodera spp. . Potato cyst nematodes . Solanum sisymbriifolium . Trap crop
Introduction Potato cyst nematodes (PCN), Globodera rostochiensis (Wollenweber 1923) Behrens 1975 and G. pallida (Stone 1973) Behrens 1975, are responsible for severe losses worldwide in potato production (Skantar et al. 2007; Watts et al. 2014; Wood et al. 2014). Potato cyst nematodes are present in almost all countries where potatoes are grown, being considered quarantine species (EPPO 2013, 2017). They have a great economic impact on seed potato producing countries and have been detected in at least 65 countries, including Portugal (Cunha et al. 2004, 2012). The first report of PCN in Portugal dates back to 1956 in the Bragança area. However, the introduction was likely prior to this date since the nematodes had already dispersed into cropland. All populations were identified as G. rostochiensis (Macara 1963). Later,
Plant Soil
G. pallida was found in fields of the Trás-os-Montes and Alto Douro area of the country where the potato production is higher (Santos and Fernandes 1988; Santos et al. 1995). In Portugal, the potato is one of the most important agricultural crops (1 million tonnes/year) with a per capita consumption of 108 kg/annum. It is estimated that an average of 40,000 ha of potato plants are grown every year. The Portuguese average potato yield is much lower than in the rest of Europe and this is partly due to the presence of PCN in cultivated fields (Cunha et al. 2012), which in some cases can cause 100% damage (Cunha et al. 2004, 2012). The most abundant nematode species in Portugal is G. rostochiensis, although there are also populations of G. pallida (Cunha et al. 2004). As PCN may be one of the main causes of the decline in potato production in Portugal, the application of control measures to reduce PCN populations present in fields is an urgent matter. Their control is difficult given the nature of these nematodes, which in the first stages of life lie within a cyst, protected from harsh conditions and able to survive in the soil for many years in the absence of host plants (Marks and Brodie 1998). The use of chemical nematicides is an effective control strategy but European legislation (Directive 69/465/ CEE (1969); Directive 2009/128/EC) is very strict regarding the use of nematicides on European soil, mainly focusing on environmental safety issues and health risks (Renčo et al. 2014). Under optimal conditions, fumigants can reduce populations of PCN by up to 80%, with the advantage of allowing short periods of crop rotation. Nevertheless, in practice, the use of these nematicides has a high cost/benefit ratio (Schomaker and Been 1999). Crop rotation with species that are not susceptible to the nematode is another of the strategies used. This practice alone is not profitable for farmers due to the lengthy rotations that are required for the PCN populations to decline to non-threatening levels (Cunha et al. 2012; Wood et al. 2014). The use of potato cultivars resistant to PCN has been used as a control strategy, thereby alleviating the need for chemical nematicides (Kaplan and Keen 1980; Roberts 1992; Williamson and Hussey 1996), but this measure cannot, so far, be considered permanent due to the existence of populations capable of overcoming the resistance that has been found (Roberts 1992; Timmermans 2005). The mix of species in the soil also hampers the success of resistant cultivars, since none are resistant to both
species of PCN and because G. pallida is more difficult to control due to the greater heterogeneity of its populations and the increased numbers of virulence groups (Scholte 2000a; Timmermans 2005; Sasaki-Crawley et al. 2010). Currently, other control alternatives are being developed, including the use of trap crops (Scholte 2000b; Timmermans 2005; Holgado and Magnusson 2010; Sasaki-Crawley et al. 2010; Dias et al. 2012) and of biological agents such as fungi and bacteria that share the rhizosphere with the nematodes. Natural control has been detected in several soils where the build-up of natural enemies, under some perennial crops and under those grown in monocultures, leads to the control of cyst nematodes, Globodera spp. and Heterodera spp., and root-knot nematodes, Meloidogyne spp. (reviewed in Stirling 2011). The majority of the natural enemies found to parasitise nematode hosts in suppressive soils, i.e. soils that contain microbial communities able to prevent the increase of nematode populations on susceptible crops, are bacteria and nematophagous fungi (Viaene et al. 2006). According to Scholte (2000b, 2000c) and Timmermans (2005), that tested the plant in the field, Solanum sisymbriifolium Lamarck (Solanaceae) proved to be a good trap-crop against PCN since its root exudates promote the hatching of second-stage juveniles (J2) without allowing the reproduction of either species of PCN, and because it works well in temperate climates such as those found in Europe. It is also resistant to the night frosts of autumn, although it will not withstand harsh winters (Scholte 2000b). Not being a plant native to Europe, the risk of introduction into the local flora should be analysed. Studies have shown that S. sisymbriifolium is a poor or very poor host of the causative agents of disease affecting the Solanaceae and should not be seen as a threat to the local flora, especially those of this family (Alconero et al. 1988; Timmermans 2005). In Portugal, this species does not exist in the native flora (www.issg.org), but other plants of the same genus, and also considered invasive, namely S. linnaeanum and S. mauritianum, are present in certain regions of the country (Marchante et al. 2014). Studies have been conducted using extracts of S. sisymbriifolium in the control of other phytoparasitic nematodes, such as Pratylenchus goodeyi Sher and Allen, 1953 (Pestana et al. 2009) and Meloidogyne spp. (Dias et al. 2012), and the aqueous extracts of S. sisymbriifolium were effective in killing P. goodeyi
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(Pestana et al. 2009). Some S. sisymbriifolium cultivars were resistant to M. chitwoodi Golden et al. (1980), M. javanica Orton-Williams (1972) and M. hispanica Hirschmann (1986) and its root exudates, also tested on the emergence of these nematodes, inhibited the hatching of J2 of five isolates of Meloidogyne species (Dias et al. 2012). Here we evaluate the effect of S. sisymbriifolium in PCN populations in the field in Portugal. Our goal is to improve crop protection systems based on trap crops (i.e. S. sisymbriifolium cvs) for the control of PCN populations. This is the first report of a trap crop being used in Portugal for the management of plant-parasitic nematodes.
Materials and methods Fields Two fields located in the center of Portugal, one in Aveiro District (Barcouço village) and another in Coimbra District (Bolho village) were selected not only due to the large number of cysts in the fields and the species identification results, G. rostochiensis and G. pallida respectively, but also due to the easy access to the fields. The Barcouço field formerly underwent a crop rotation of potatoes for several years and 1 year of cabbage, but this was a long time ago. The farmer did not know what potato cultivars were used. The Bolho field had grown potatoes (cvs Agria and Kondor, both susceptible to Globodera spp.) for the last 5 years and, before that, the farmer planted tobacco, Nicotiana tabacum L. (another solanaceous crop), for several years. Soils were characterised in relation to their physical and chemical characteristics (Table 1). Globodera isolates identification Identification of the two Globodera isolates was performed by PCR-RFLP, with specific primers and the enzymes Alu I and Hinf I (Sirca et al. 2010). DNA was extracted from single cysts from both isolates using the QIAGEN Kit (DNAeasy®Blood and Tissue Handbok). A total of 10 cysts/isolate were used and DNA amplified by PCR using the procedure described by Sirca et al. (2010). The following primers were used for amplification of the ITS-rRNA gene: forward, CGTAACAA GGTAGCTGTAG and reverse, TCCTCGCTAAATGA
Table 1 Physical and chemical characteristics of soil from Barcouço and Bolho fields Parameter
Barcouço
Fine sand (%)
82.5
87.1
Organic matter (%)
4.08a
2.07a
pH (H2O)
6.0b
P (mg/1000 g) K (mg/1000 g)
Bolho
4.7b c
> 200 d
173
> 200c 176d
≤ 1.0 (Very low); 1.1–2.0 (Low); 2.1–4.0 (Medium); 4.1–6.0 (High); > 6.0 (Very High)
a
b
≤ 4.5–6.5 (Acidic); 6.6–7.5 (Neutral); 7.6–9.5 (Alkaline)
≤ 25 (Very Low); 26–50 (Low); 51–100 (Medium); 101– 200 (High); > 200 (Very High)
c and d
TAT (Ferris et al. 1993). PCR reactions were prepared with Enzyme buffer 1× (Bioline), 1.5 mM MgCl2 (Bioline), 0.2 mM dNTP (Bioline), 0.5 μM of each primer and 10 μL of DNA in a total volume of 25 μL. Amplification was carried out in a BIO-RAD Thermal Cycler (Bio-Rad Laboratories, Inc.) using the following conditions: initial denaturation at 94°C for 2.5 min.; 35 cycles of denaturation at 94°C for 1 min, annealing at 48°C for 45 s, and elongation at 72°C during 1 min; final elongation at 72°C for 2 min. Four μL of the amplification products were incubated with 5 U of the restriction enzymes Alu I or Hinf I at 37°C during 3 h (Sirca et al. 2010). Fragments were separated on 2% agarose gel and visualised with GreenSafe (NZYTech). This procedure was repeated twice to check the reproducibility of the reactions. Globodera spp. multiplication In order to obtain a large number of cysts, the PCN isolates, G. pallida and G. rostochiensis, from Bolho and Barcouço fields respectively, were reared on susceptible potato, S. tuberosum ssp. tuberosum L. (cv. Désirée). The potato plants were inoculated with second-stage juveniles (J2) from field collected cysts placed in water during 1 week and then in root exudate of potato cv. Désirée. Root exudates were obtained through successive leaching of soil from 500 mL pots containing 1 month old plants (Shepherd 1986). The exudates were collected, filtered using Whatman filter paper and stored at 4°C. Hatched J2 were counted daily until enough inoculum had accumulated. The plants were obtained from pieces of potato tubers with sprouts, which were placed in plastic containers filled with 800 g
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of sterile sandy soil, in a glasshouse (20–25°C, 70–75% relative humidity and 12 h photoperiod). One week after planting, the plants were inoculated with 1800 J2/container. Fertilizer (Hyponex - 7% N (0.21 g/100 mL), 6% P (0.18 g/100 mL) and 19% K (0.57 g/100 mL) was added once a week and the plants were watered every day. Ten weeks after planting, the tops of the plants were removed and the soil dried. The new cysts were extracted using a Fenwick can, separated from debris and stored at 4°C (Fenwick 1940; Shepherd 1986). Plants of Solanum sisymbriifolium Plants of S. sisymbriifolium (cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001), from Vandjike Semo (now VanDinter Semo, The Netherlands), were reared continuously. The seeds of S. sisymbriifolium were placed in polystyrene plates with sterile peat in a glasshouse (20– 25°C, 70–75% relative humidity and 12 h photoperiod). After germination, the plants were removed from the plates and transferred to 500 mL pots containing a sterilised mixture of soil and sand and maintained in the same glasshouse. Similar size plants were chosen to extract the exudates (3 months old) and to be inoculated (1 month old) with J2. The exudates were extracted 3 months after the plants were transferred to pots when they had a well-developed root system. Effect of Solanum sisymbriifolium on hatching of Portuguese PCN populations in the laboratory Cysts collected directly from fields, extracted and not allowed to dry, and cysts obtained in the laboratory (1st generation) were used in the assay. Solanum sisymbriifolium (cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001) and S. tuberosum cv. Désirée root exudates were obtained as described above. Potato (S. tuberosum cv. Désirée) root exudate and tap water were used as controls. Fifteen cysts of each PCN isolate were placed inside sieves, in Petri dishes, with tap water during 1 week. The water was then replaced by 5 mL of root exudate of each plant cultivar. Five replicates/treatment were used. The experiments were conducted in the dark in a humid chamber at a temperature of 22 ± 2°C. The number of emerged J2 were recorded daily over a maximum period of 30 days. The root exudates were replaced by fresh exudate daily. After 30 days, the cysts were crushed with a glass tube on an aluminium plate with a slight depression to release the J2 and the number of J2 remaining were counted. The
suspension was transferred to a plastic cup and stirred vigorously in water using an electrical stirrer to separate the J2 from the cysts. Three replicate of 1 mL subsamples of the egg suspension were transferred to counting slides and the J2 quantified. The mean number of J2 in each mL of suspension was calculated. The cumulative hatching percentage (CH%) was determined. The experiment with cysts from the laboratory was repeated twice and the results were similar. Reproduction of PCN on Solanum sisymbriifolium cultivars The resistance/susceptibility of S. sisymbriifolium cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001 against the PCN isolates was assessed. The pathogenicity of the isolates of each species of Globodera was evaluated on five plants of each S. sisymbriifolium cultivar inoculated with 1800 J2/plant and S. tuberosum cv. Désirée was used as control. The plants were placed in plastic containers filled with sterile sandy soil, in a glasshouse (20–25°C, 70–75% relative humidity and 12 h photoperiod). Fertiliser (Hyponex - 7% N, 6% P and 19% K at the same rate mentioned above) was added once a week and the plants were watered every day. Ten weeks after inoculation the tops of the plants were removed, the soil dried at room temperature, the cysts extracted using a Fenwick can, separated from debris and counted and the number of J2 inside the cysts estimated using the procedure described above. Based on the number of cysts and J2/cyst, the final population density (Pf) was calculated. The reproduction factor (Rf = Pf/Pi), where Pi is the initial population density and Pf the final population density, was also determined (Oostenbrink 1966). Effects of Solanum sisymbriifolium on Portuguese PCN population densities in the field The experimental sites in both fields (Barcouço and Bolho) were cropped with potatoes (2012) and divided into 21 plots (5 m2/plot). All plots were sampled to assess the number of cysts and J2 in the soil (Pi). The S. sisymbriifolium cvs (three replicates/cv) were then seeded in plots, randomly distributed, (Fig. 1) according to the parameters referred in Table 2. This was done in 2012 and repeated in 2013. The number of plants/plot in Barcouço in 2013 was very low and the experiment was repeated in 2014/2015 only in this field. During March, 2012 and April, 2013, tubers of S. tuberosum cv. Désirée were planted in both fields
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Fig. 1 Scheme performed in both Barcouço and Bolho fields. P/ Melody (Potato/Solanum sisymbriifolium cv. Melody); P/Pion (Potato/S. sisymbriifolium cv. Pion); P/Sharp (Potato/ S. sisymbriifolium cv. Sh arp); P/Sis 4004 (Potato/
S. sisymbriifolium cv. Sis 4004); P/Sis 6001 (Potato/ S. sisymbriifolium cv. Sis 6001); P/F (Potato/Fallow – or Positive control) and F/F (Fallow/Fallow – negative control). Twenty-one plots (5 m2 each) with three replicates/cultivar
(Barcouço and Bolho) and during April 2014 only in Barcouço field. On the day of planting, a fertiliser dressing (133 Kg N, 133 Kg P2O5 and 133 Kg K2O) was applied in each field. During May 2012 and 2013, a top-dressing (225 kg nitrogen) was given during the growing season and weeding-hilling was carried out along with phytosanitary treatments against mildew (Propineb) and Colorado beetle (chlorpyrifos). At the end of the growing seasons, all plots were sampled to assess the number of cysts and J2 in the soil. During July of 2012 and September of 2013, the S. sisymbriifolium cultivars were sown, in both fields, and in June 2015 only in Barcouço field. The plants were grown for 6 months and destroyed in January of 2013, March of 2014 and December of 2015. The fields were then sampled again to assess the number of cysts and eggs/J2 in the soil (Pf) (Table 3). During each year of the assay there was a crop of potatoes that rotate with one crop of
S. sisymbriifolium. Soil samples were collected before planting and after harvest of each crop. Three months after planting, the potato crops were harvested and the yields determined in each plot (weight of new tubers from five plants/plot) in both fields. Flowers of S. sisymbriifolium plants were cut in order to avoid fruit formation and any potential spread of the plants through seed dispersal. All plants were destroyed after 6 months in the field and incorporated into the soil. Soil samples were collected from each plot, prior to planting and after harvesting, and three sub-samples (500 cm3) randomly taken were used for cyst extraction and the cysts counted. From each sample, 50 cysts were randomly selected and placed in a glass block containing tap water during 24 h. After this period, the J2 and eggs were extracted from cysts and quantified as described before. When there were less than 20 cysts, the J2 and eggs were not counted. The number of J2 was considered too low and not significant in relation with infestation levels.
Table 2 Parameters of the plots sown with Solanum sisymbriifolium cultivars in Barcouço and Bolho fields Parameter
Barcouço
Bolho
Seed density (seeds/m2)
400
400
Number of rows
13
15
Row length (m)
3
3
Distance between rows (cm)
13
13
Seed spacing in the rows (cm)
2
2
Statistical analysis Cumulative J2 hatching percentage (CH%) from field and laboratory cysts for both Globodera species and potato yield from both fields were statistically analysed, using One Way Anova and a LSD at the 5% level test (Statistica 10). An x^1/3
Plant Soil Table 3 Chronology of events in the years 2012–2015 in Bolho and Barcouço fields Solanum species
Sowing
Harvest/destruction
2012
2013
2014*
2015*
2012
2013
2014*
2015*
S. tuberosum
March
April
April
_____
July
July
July
____
S. sisymbriifolium
July
Sept.
____
June
_____
Jan.
March
Dec.
*Only in Barcouço
transformation was used to ensure a normal distribution and homogeneity of variance of the data. The values obtained for cyst and egg counts from the field were statistically analysed using a Kruskal-Wallis test with a posteriori multiple comparisons whenever statistically significant differences were detected between at least one pair of cultivars (program SPSS, version 22).
Results Globodera isolates identification The patterns obtained were similar to those characteristic of G. rostochiensis (Barcouço field) and of G. pallida (Bolho field) (Fig. 2).
Effects of Solanum sisymbriifolium on hatching of Portuguese PCN populations The CH% for all cultivars and controls for the field cysts (Fig. 3) was higher for cysts obtained in the laboratory (1st generation, more than 6 months old) (Fig. 4). The effect was higher in the positive control (S. tuberosum cv. Désirée) while in the negative control (water) was lower. Root exudates from all S. sisymbriifolium cvs had a positive effect on the hatching of J2, for field cysts, with the values higher for G. rostochiensis but lower when compared with the positive control (S. tuberosum cv. Désirée). The hatching was always higher than in water but lower than in potato root exudate. With the exception of the negative control (water), in all treatments the hatching effect was higher for G. ro stochien sis than for G. pallida. With
Fig. 2 PCR-RFLP patterns of the Globodera pallida and G. rostochiensis isolates, in agarose gel, obtained with the restriction enzymes Alu I (AI) and Hinf I (HI). M – Molecular marker (HyperLadder IV 100 bp)
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Fig. 3 Cumulative hatching percentage of second-stage juveniles from field cysts of Portuguese potato cyst nematodes, Globodera rostochiensis (Barcouço) and G. pallida (Bolho), for 30 days in the presence of Solanum sisymbriifolium root exudates (cvs Melody,
Pion, Sharp, Sis 4004 and Sis 6001). Solanum tuberosum (cv. Désirée) and water were used as controls. Columns with the same letter are not significantly different according to LSD test (p < 0.05)
G. rostochiensis the higher values were obtained with cvs Melody (49.6%) and Sharp (45.8%) followed by cv. Sis 4004 (42.5%). With G. pallida the higher values were obtained for cvs Sharp (19.8%) and Pion (17.9%). For laboratory cysts, the root exudates from Sis 6001, Melody and Pion had a positive effect on the hatching of PCN J2 for both species and the root exudates from Sis 6001 had a positive effect on the hatching of PCN J2 for G. pallida (statistically similar between them). The lowest hatching effect observed in both PCN species was with cv. Sharp (Fig. 4), whose values were similar to those obtained in water. The highest values for the J2 hatching effect were observed with cv. Sis 6001 (13.7% for G. rostochiensis and 7.8% for G. pallida) although
they were lower than with the potato exudates (24.4% for G. rostochiensis and 37.0% for G. pallida). The highest value obtained with potato exudate was around 70% for field cysts (G. rostochiensis) and 37% for laboratory cysts (G. pallida) (Figs. 3 and 4).
Fig. 4 Cumulative hatching percentage of second-stage juveniles from laboratory cysts of Portuguese potato cyst nematodes, Globodera rostochiensis (Barcouço) and G. pallida (Bolho), for 30 days in the presence of Solanum sisymbriifolium root exudates
(cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001). Solanum tuberosum (cv. Désirée) and water were used as controls. Columns with the same letter are not significantly different according to LSD test (p < 0.05)
Reproduction of PCN on Solanum sisymbriifolium cultivars The length of the experiments (10 weeks) and the environmental conditions proved to be sufficient and suitable for the development and reproduction of the nematodes. The values of number of cysts (NC) and Pf o b t a i n e d o n S . t u b e ro s u m c v. D é s i r é e f o r
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G. rostochiensis (192 and 23,294 J2 respectively) confirmed the viability of the inoculum and that environmental conditions were favorable for penetration, development and reproduction of nematodes. For G. pallida, although the nematode did not reproduce on S. sisymbriifolium, the values obtained on potato were not as high (Rf = 2.0) as for G. rostochiensis (Rf = 13), indicating that the inoculum was not in very good condition or that the nematodes were in diapause. Both PCN populations did not reproduce in none of the S. sisymbriifolium cultivars. All cultivars of this plant were resistant to the PCN populations.
Effects of Solanum sisymbriifolium cultivars on Portuguese PCN population densities in the field In Barcouço, after 1 year of S. sisymbriifolium growth, the yield in 2013 was almost the double of the the yield of 2012. The yield of potatoes in 2014 (Barcouço) was not considered because the production was simply equal to the weight of potatoes that were planted (25 Kg in all plots) (Fig. 5). In the Bolho field, in 2012, the seeds of S. sisymbriifolium did not germinate because the temperature was too high and the irrigation was insufficient for the conditions. The field was again cleared of weeds
and the sowing of S. sisymbriifolium cvs repeated. This time, the temperature was lower, watering was controlled and germination was obtained. In 2013, the S. sisymbriifolium cvs grew very well in the Bolho field and in some plots there were more than 100 plants. In the Barcouço field, in 2012, the plants grew very well but in 2013, due to a long period of rain, weeds suddenly emerged when the plants were starting to grow and only a few S. sisymbriifolium germinated in each plot. The experiment was repeated in 2014 only for Barcouço field. The number of cysts and eggs (Pi/Pf) revealed great variation between the treatments and within treatment with no statistical differences between treatments (Figs. 6 and 7).
Discussion All S. sisymbriifolium cultivars were resistant to G. rostochiensis and G. pallida populations in Portugal and promoted J2 hatching. They can be included in crop rotations and are potential candidates to incorporate in management programs against PCN. Although there were no statistical differences it seems that fallow could be the best strategy to reduce PCN populations faster
Fig. 5 Potato, Solanum tuberosum cv. Désirée, yield in the years 2012–2013 in the fields of Bolho (a) (F = 1.23, p > 0.05) and Barcouço (b) (F = 6.74, p < 0.05). Columns with the same letter are not significantly different according to LSD test (p < 0.05)
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Fig. 6 Number of cysts/100 cm 3 of soil in Solanum sisymbriifolium cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001 in Bolho (F = 9.87, p > 0.05) and Barcouço (F = 1.78, p > 0.05) fields in the beginning (Pi-2012) and at the end (Pf-2013 in Bolho
and 2014 in Barcouço) of the assay. There were no statistical differences between treatments. Pi, Initial population density; Pf, Final population density
and this avoids the introduction of an exotic plant that could become a problem. The effects of root exudates from different cultivars of S. sisymbriifolium on J2 hatching from field cysts were similar to those obtained by Timmermans et al. (2006) using G. pallida cysts but higher than those
reported by Scholte (2000b) for both nematode species that used cysts multiplied in Laboratory. Although the percentage of hatching from cysts from the laboratory was much lower, the trends were similar to those from the field. In the experiment with cysts, obtained in the laboratory, the low hatch may have been caused by cyst
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Fig. 7 Number of second-stage juveniles (J2)/100 cm3 of soil in Solanum sisymbriifolium cvs Melody, Pion, Sharp, Sis 4004 and Sis 6001 in Bolho (F = 8.43, p > 0.05) and Barcouço (F = 0.45, p > 0.05) fields in the beginning (Pi-2012) and at the end (Pf-2013
in Bolho and 2014 in Barcouço) of the assay. There were no statistical differences between treatments. Pi, Initial population density; Pf, Final population density
handling, although the cysts were extracted only with water, hand-picked and stored at 4°C. Janssen et al. (1987) remarked that when cysts were desiccated the hatchability dropped from 40 to 3%. The response of G. rostochiensis to the exudates was higher than or equal to
the G. pallida response, except for the effect of the potato exudate, although the differences were not statistically significant. The highest differences were detected in cvs Sharp and Sis 4004 exudates where there was almost no hatching from the laboratory cysts. This may
Plant Soil
be explained by the variability of the cyst contents. Nonetheless, there is a species dependent difference in the J2 response to S. sisymbriifolium exudates G. rostochiensis seems to be more stimulated than G. pallida, which is confirmed by the positive control, where they responded equally. Our values were much lower than those reported by Scholte and Vos (2000), however differences in experimental conditions could explain the disparity. The variability obtained with cysts of different ages is a factor to be taken into account when looking for reproducible results between different laboratories. All S. sisymbriifolium cvs were resistant to both species of PCN and similar results were achieved by Roberts and Stone (1983) and by Scholte (2000b), although for different cultivars. Consequently, S. sisymbriifolium can be included in crop rotation programmes against these nematodes in Portugal. Nevertheless, pathogenicity assays should be performed with local PCN populations, under field conditions, due to the large physiological variability that is associated with the PCN virulence groups. In addition, environmental conditions may affect the physiology of the plant or of the nematode and change the feedback of these cultivars to the nematode. Field trials were performed for 2 years in two PCN infested fields (Bolho and Barcouço) using the following rotation: S. tuberosum cv. Désirée followed by S. sisymbriifolium. In both fields, all cultivars had the same behaviour with no statistical differences. However, with cv. Sharp there was a slight increase of PCN J2. This is likely due to less mature plants in some of the plots where this cultivar was planted. The lower Pi in the Barcouço field can be explained by the fact that potatoes were not the main crop in this field. The field was used for other crops or it was not used at all. The cysts were damaged and the eggs had fungi, such as Pochonia chlamydosporia, that are pathogenic to PCN. In the Bolho field, potatoes were planted every year and allowed a high population density of PCN. The soils were also different in terms of pH and percentage of organic matter. The Barcouço soil was less acidic and had a higher percentage of organic matter than the Bolho soil. The yearly fertilisation of the soil, where potatoes are grown intensively, may explain the differences in pH since successive fertilisations with sulphates can lower the soil pH. In the Barcouço field, the soil was not cultivated for several years. Many weeds flourished and died and this contributed to the
higher percentage of organic matter in this field. Although the potato crop prefers slightly acidic soils (pH between 5.0 and 6.0) it is fairly tolerant. The pH in both fields was good for potato growth, although a lower pH seems to increase S. sisymbriifolium and S. tuberosum yields (Scholte 2000c). The potato yields were more affected by climatic conditions than by PCN infestations. In 2013, the yield in Bolho was lower than in 2012, probably due to the high densities of PCN populations. In contrast, the yield in Barcouço was higher in 2013. The densities of PCN population were much lower than in the Bolho field. Additionally, S. sisymbriifolium cvs grew much better in 2012 in this field than in Bolho, consequently reducing the PCN population densities and allowing a better yield. The pH and organic matter were also better for potato growth in Barcouço. The intensive repeated cropping of potato in Bolho typically leads to a reduction in yield in ensuing years and 2014 was not good for potatoes in Portugal due to high rainfall during the growing season. When comparing the two fields, the variability of the results was much bigger in Barcouço and that can be explained by the homogeneity (number of cysts, health of the PCN populations and field topography) found in Bolho plots. Although the climatic conditions were the same because the distance between fields is not significant, some plots in Barcouço had trees that shaded the soil and thereby maintained humidity, others plots were always exposed to the sun and other had a slope that encouraged the soil to dry faster. Another important aspect is that the PCN species in Barcouço and Bolho fields reacted differently to the plant exudates confirming the results obtained in the laboratory experiments. The reduction of J2 of G. pallida (Bolho) is in accordance with other studies where a reduction of ~80% due to the planting of S. sisymbriifolium was achieved and revealed no statistical differences among cultivars (Scholte and Vos 2000; Hartsema et al. 2005; Timmermans et al. 2006). The reduction of G. rostochiensis J2 (Barcouço) was similar for cvs Melody and Sis 6001, less for cvs Pion and Sis 4004, and there was a slight increase of J2 where cv. Sharp was sown. The greatest reduction in J2 numbers was observed in the fallow plots. According to Scholte (2000c), the reduction of PCN decreased with increasing soil infestation, which is not in agreement with our results. The Bolho field had a much higher infestation
Plant Soil
than Barcouço and, at the end of the experiment, the reduction was relatively higher in this field. Although there was a decrease in the PCN population densities at the end of the experiments, the differences among treatments were not statistically different. Nonetheless, there was a reduction of the nematode populations in both fields in the plots where S. sisymbriifolium was grown when comparing with the plots where the crop rotation was potato/fallow. Similar results were obtained when only fallow was used. However, the use of fallow is not profitable for the farmers. Dandurand and Knudsen (2016) observed that S. sisymbriifollium had a greater effect on the reduction of a G. pallida population density than fallow. The differences can be due to the experimental design. When testing the effects of living organisms in the field there is always other variables that cannot be controlled. It can also be concluded that, in our climatic conditions, a high reduction of the PCN population densities were obtained in 2 years of the proposed cropping scheme. This should be taken into account when deciding on possible quarantine measures. The decline/year of a PCN population in cold climates will be much lower than in hot climates such as those from Mediterranean countries. Even so, cvs Melody and Sis 4004 were the most successful trap crops to improve the control of PCN populations in potato fields. They are resistant to PCN, the root exudates have hatching factors for both PCN species, and reduce population densities to lower levels. Those cultivars should be used with care and only when root-knot nematodes (RKN), Meloidogyne spp., are not detected coexisting with PCN. Our studies (Dias et al. 2012) revealed that cv. Melody was susceptible to M. arenaria and M. hapla and hypersusceptible to M. hispanica; cv. Sis 4004 was susceptible to M. hapla, M. hispanica and hypersusceptible to M. arenaria. Both cultivars were resistant to M. chitwoodi and M. javanica. Scholte and Vos (2000) stated that S. sisymbriifolium cvs were resistant to RKN. An important note for the use of S. sisymbriifolium for the management of PCN in Portugal is the need to cut the upper part (flowers and fruits) before the seed production to prevent its dispersal. This procedure does not affect root growth and the shoots continue to develop. In this study, we demonstrated that these trap crops can reduce the PCN population densities. Using a trap crop does not disturb the ecological balance in the soil and their roots grow deeper into the soil than
nematicides can penetrate. The incorporation of the plants in the soil can also be a source of nutrients and organic matter and the substances released may act as biofumigants, which can increase the effect of the trap crop on PCN. In our climatic conditions, the use of a mixture of S. sisymbriifolium cvs could improve the efficacy of integrated pest management programs for PCN control. Our study also suggests that incorporating a fallow period in a crop rotation scheme with a nonhost crop is a management strategy that can reduce PCN population densities and decrease the time for crop rotation required by EU directives. Acknowledgements This research was partially supported by the European Regional Development Fund (FEDER) through the BPrograma Operacional Factores de Competitividade (COMPETE) and by National funds through Fundação para Ciência e a Tecnologia^ (FCT) under the project PTDC/101817/ 2008. Thanks are also due to Vandijke Semo, Scheemda, The Netherlands, for supplying Solanum sisymbriifolium seeds and to the farmers, Mr. Mário Coelho (Barcouço) and Mr. Antero Ferreira (Bolho), who provided the fields for the assays and helped with the irrigation and field treatments.
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