Evaluation of repeated biodisinfestation using Brassica carinata pellets to control Meloidogyne incognita in protected pepper crops

The nematode Meloidogyne incognita is responsible for substantial losses in greenhouse-grown peppers in southeastern Spain. This study evaluates the use of biodisinfestation (BS) (organic amendment + solarisation) as an alternative to using methyl bromide (MB) over three consecutive years to control the nematode in greenhouse conditions. Brassica carinata (BP) pellets or B. carinata (BP) + fresh sheep manure (M) were evaluated in treatments which began on two different dates (August and October) and the results were compared with MB-disinfested and untreated controls. During the third year, the gall index for BP was lower than that obtained for BP + M and in the August treatment than in the October treatment. The commercial crop of pepper fruit obtained with the biodisinfestation treatments begun in August was similar to or higher than that obtained with MB, and higher than that obtained with both October biodisinfestation treatments. The yield of the October biodisinfestation treatments was higher than that of the untreated one. In August of all the years studied, the accumulated exposure times were greater than the thresholds required to kill M. incognita populations at 15 cm depth. The incidence of the nematode did not correspond to the reduction achieved during solarisation, and seemed to increase during the crop cycle. Further studies should look at why high temperatures do not produce a sustained reduction in the populations of Meloidogyne incognita. Additional key words: greenhouses; nematodes; organic amendments; soil. * Corresponding author: mariam.guerrero@carm.es Received: 27-06-12. Accepted: 04-04-13. Abbreviations used: BP (Brassica carinata pellets); BS (biodisinfestation); GI (galling index); M (fresh sheep manure); MB (methyl bromide); VIF (virtually impermeable film). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2013 11(2), 485-493 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2013112-3275 eISSN: 2171-9292


Introduction
In the province of Murcia (south-eastern Spain) pepper (Capsicum annuum L.), is a crop that occupies more than 90% of the area dedicated to greenhouse crops and has been a monoculture for the last 20 years (Lacasa & Guirao, 1997).The normal crop cycle lasts 9-10 months (November-December to September-October).Phytophthora spp.(P.capsici and P. parasitica) and Meloidogyne incognita are recurring and persistent problems and, together, cause substantial crop spoilage (Tello & Lacasa, 1997;Bello et al., 2004).
Until 2005, methyl bromide (MB) was used to disinfect soils to control both pathogens and to reduce the effects of fatigue caused by repeated monocultures (Martínez et al., 2011).Since that time, MB has been replaced by a mixture of 1,3-dichloropropene and chloropicrin, but these, too, will shortly be banned by EU legislation.To this forthcoming ban must be added the growth in demand for ecological foods produced by sustainable agricultural practices, meaning that nonchemical methods must be sought for controlling soilborne plant pathogens and plant parasite nematodes adapted for use in intensive horticulture.
In recent years numerous alternatives to chemical disinfection have been studied, and of these, those based on organic amendments alone or in combination with solarisation seem to be the most promising ones.This method involves incorporating decomposable organic matter into the soil and subsequent humidification, covering with plastic for 2-15 weeks.Decomposition of the source of carbon in the humid or wet soil by anaerobic microorganisms reduces the oxygen content below the plastic (Messiha et al., 2007) in a process that has been called "biological disinfestation" (Blok et al., 2000), "reductive soil disinfestation" (Katase et al., 2009) or "soil reductive sterilization" (Yossen et al., 2008).The anaerobic conditions increase the activities of the microorganisms in the soil, which have a suppressive effect on pathogens and parasites (Gamliel et al., 2000;Bonanomi et al., 2007;Oka, 2010;Klein et al., 2011).
The combined action of organic amendment and solarisation has demonstrated its effectiveness in the control of Phytophthora capsici in pepper crops.The process of soil disinfection was called "biodisinfestation" by Núñez-Zofio et al. (2011).In these processes, the effects of anaerobiosis and suppression are added to the effect of temperature and of the gases released during decomposition of the organic matter, such as ammonia and nitrous acid (Lazarovits, 2001;Tenuta & Lazarovits, 2002;Arriaga et al., 2011).Although the characteristics of the OM are regarded as critical elements for the control of pathogens in general, the same characteristics do not seem to have a great impact on the control of some pathogens produced in anaerobic conditions (Bonanomi et al., 2007(Bonanomi et al., , 2010)).
The use of Brassicaceae in crop rotations or as green manure amendment in biofumigation treatments (Stapleton & Bañuelos, 2009) has been seen to reduce the incidence of some soilborne pathogens and plant parasites, including nematodes (Smolinska et al., 2003;Larkin & Griffin, 2008) through their release of isothiocyanates.For example, improved pathogen and weed control has been achieved by using amendments obtained from by-products produced during the extraction of oil from B. carinata and Sinapis alba seeds (Lazzeri & Manici, 2000;Lazzeri et al., 2004;Cohen et al., 2005;Palmieri, 2005;Sachi et al., 2005).
Biodisinfestation (BS) using fresh sheep manure (M) as amendment in August was seen to provide similar results to those obtained using MB to control Phytophthora spp. in pepper crops (Guerrero et al., 2004a).Production increased when the application was repeated more than two years (Guerrero et al., 2004b(Guerrero et al., , 2006;;Candido et al., 2005), but it seemed to have little effect when applied after the beginning of September (Guerrero et al., 2010).
Some growers are reluctant to use BS because they should be able to shorten the regular cycle by one or two months, which implies a substantial reduction in the crop yield (20-30%).The implementation of BS is one of the drawbacks of the method because BS must begin in August (Guerrero et al., 2004a).
The objective of the present study was to evaluate the effect of BS using B. carinata pellets (BP) alone or combined with M, when applied in August or October for the control of Meloidogyne incognita as well as marketable yield in greenhouse grown pepper crops in a mild-warm climate.The experiment was carried out over three consecutive years and the treatments were repeated in the same plots.
The soil was naturally contaminated by M. incognita (race 2, biovar pepper according to Robertson et al., 2006).The experiment treatments were arranged in a completed randomized block design with three replicates, and a plot size of 52 m 2 .The soil was a clay loam with an organic matter content of 2%, a total nitrogen content of 1.5 g kg -1 , pH = 7.8, a C/N ratio of 10.3 and electrical conductivity (EC) of 14.4 dS m -1 .
Two treatments were studied: (i) BP: solarisation + B. carinata pellets (0.3 kg m -2 as organic amend-ment); (ii) BP + M: solarization + a mixture of seed meal B. carinata pellets (0.3 kg m -2 ) and fresh sheep manure (2.5 kg m -2 ).Treatments were carried out for 6 weeks in both August and October (August and October treatments were performed in different experimental plots).Untreated (neither solarisation nor organic amendment) and MBtreated plots were included as reference controls.
Organic amendments were buried using a rotary tiller and a drip irrigation system (3 L h -1 emitters spaced 0.40 m apart in the same rows) was installed.Then, a transparent plastic (polyethylene, 0.05 mm) was spread over the BS treatments plots and the soil was watered (up to 25-30 cm) for 3 h day -1 on two consecutive days.The B. carinata pellets (BP) (Biofence Triumph Italia SPA) contained 6% N, 7% P, 2.4% K and 84.2% organic matter.The glucosinolate sinigrin constituted the B. carinata seed meal as well as the derived isothiocyanates which are effective against Meloidogyne nematodes (Lazzeri et al., 2010).The applied manure had the following characteristics: pH 8.2 and EC 5.0 dS m -1 , organic C: 400 g kg -1 , total N: 25.0 g kg -1 , total P: 5.5 g kg -1 and total K: 26.1 g kg -1 .
In the treatments not including M, composted manure was added at 2.5 kg m -2 before planting.MB (98% a.i., 2% chlorpicrin) was applied at a dosage of 30 g m -2 as a cold fumigation, using a volumetric dispenser after the soil was covered with virtually impermeable film (VIF, 0.04 mm thick) and maintained for one week.
In January, pepper plants were planted with a crop density of 25,000 plants ha -1 corresponding to 3 rows of 45 plants per row.The crop season ended in the first week of August.Pests were controlled according to Integrated Pest Management (CAARM, 2010).

Parameters studied
Soil temperature (at 15 and 30 cm depth) was monitored using sensors connected to a data-logger (H8-4 32K, Hobo Weather Stat., Onset Comp.Corp., Cape Cod, MA, USA) with continuous data recording and reading every 15 min.
Pepper fruits were harvested throughout the crop season and final marketable yield (kg m -2 ) was determined.
The incidence of M. incognita was evaluated at the end of the crop season.Ten pepper plants of each experimental plot were pulled up in order to determine (i) the percentage of galled plants and (ii) the galling index according to Bridge & Page (1980) on a scale of 0-10.

Statistical analyses
The data were studied using ANOVA, Statgraphics Centurion XV.II.Differences among treatments were tested using the LSD test al 95% (p < 0.05).The data were transformed to achieve homoscedasticity and normality using the following transformations: log (x + 1) for the crop yield and nodulation index, and the formula arcsine ͌ʳʳ (x) for the percentage of galled plants.

Incidence of Meloidogyne incognita
In the first year the galling index (GI) of BS (BP and BP + M) was smaller than in the untreated control and the starting date of the treatment or amendment had no influence (Table 1).In the second year the GI of the August BS treatments was lower than that of the October BS treatments finding no differences between amendments in August, but in October there were differences.
In the third year there were differences in the GI between dates (August and October) and between both amendments.The GI of the October BP + M did not differ from that of the untreated control.The incidence was lower in the BP treatments than in the BP + M in both dates.The GI values of the August treatments were more similar to those of MB than those of the October treatments.The BP treatment beginning in August reduced the incidence of Meloidogyne in the third year of application (GI was 3.8 in the first year, and 2.6 in the third year), being more pronounced the reduction in the treatment with BP (31.58%) than with BP + M (14.6%).In the October treatments the incidence of M. incognita increased (50% in BP + M and 31% in BP), and also in the non-treated control (20%) and MB (150%).
The percentage of infested plants varied very little between the BS treatments and the non-treated control  in all three years.However, this proportion increased in MB in the last year.In the August BS treatments there was a non-significant tendency for the percentage of infested plants to fall as time passed.In the plots with GI values below 4 no symptoms of yellowing, reduced growth, etc, were evident in the aerial part of the plants.

Effect on pepper crop yield
In the first year, differences were observed between BS dates but not between amendments, within a same date, being higher in August than in October (Table 2).Yield for the October treatments were similar to those obtained with MB but higher than those obtained in the non-treated control and lower than in the August BS treatments.
In the second year, there were no differences between dates or between both amendments (August and October) (Table 2), the production obtained with BS treatments being similar to that obtained with MB and higher than in the control.
In the third year, there were differences between dates but not between amendments (Table 2).The production obtained in October was lower than that obtained with MB, which was similar to that obtained in August.The production obtained with BS at any date was higher than that obtained with the control, as in the other years.
The August BS treatments provided similar levels of production or higher than MB every year, while the October BS treatment yields were no different from MB in two of the three years and always higher than the non-treated control.

Soil temperatures
In general, the daily evolution of the maximum, mean and minimum temperatures was similar for the same depth (15 and 30 cm) and treatment date (August and October).
Differences between depths and also between dates for the same treatment were observed and between the treatments and the untreated control.Table 3 depict the percentage of the total BS time that the soil temperatures were in given ranges above 30°C.Above 38°C the values are represented at 1°C intervals, as suggested by Wang & McSorley (2008).
In the August treatments the temperature at 15 cm always were above 25°C the first year, above 30°C the second year and above 32°C the third year, while at 30 cm the corresponding minimum temperatures were 28°C, 32°C, and 34°C, respectively.In the first year the percentage of time that the soil at 15 cm was above 42°C was higher for BP + M (24.44%) than for BP (0.3%), while at 30 cm the differences between the amendments were slight (0.6% for BP and 0.10% for BP + M).In the second year the percentage of differences between soils with temperatures above 42ºC were very small at 15 cm (1.12% for BP and 3.70% for BP + M), while at 30 cm the temperatures did not exceed 42°C in either amendment.In the third year the soil temperature frequently exceeded 42°C at 15 cm with both treatments (18.70% for BP, 23.28 % for BP + M) while at 30 cm the corresponding times were BP + M (22.41%) and BP (1.65%).
The first year in October treatments the temperature was always lower than 38ºC at both depths and for both amendments except the second year at 15 cm for BP, where the soil was above this temperature for 8.9% of the time.The percentage of time when the soil was above 35°C was greater for BP + M than for BP at both depths the first year (17.07%and 3.5%, respectively, at 15 cm and 21.2% and 6.8%, respectively, at 30 cm).In the second year, the time with temperatures above 35°C was similar for both amendments at 15 cm (16.6% for BP + M and 25.3% for BP) at 30 cm (16.39% for BP + M and 23.58% for BP).In the third year, only the soils in BP + M at 15 cm exceeded 35°C (4.40% of the time).
In the untreated control the temperature was lower than for both treatments at both depths and dates during the three years of the experiment.Only in August of the first year did the soil temperature rise above 38°C (1.93% of the time).

Discussion
In previous studies, we showed that BS with fresh sheep or poultry manure provided acceptable levels  of M. incognita control (Guerrero et al., 2004a(Guerrero et al., , 2006) ) which improved as the treatments were prolonged over 3 to 7 years (Guerrero et al., 2007).The results of the present study show that BS with seed mail B. carinata pellets, alone or in combination with fresh sheep manure, provides effective control of M. incognita when begun in August and ensures similar production levels to those obtained using MB.However this treatment was not effective when begun in October.
Many species of the family Brassicaceae contain glucosinolates, which upon decomposition, give rise to isothiocyanates (Smolinska et al., 1997;Kirkegard & Sarwar, 1998), which reduce the incidence of pathogens in the soil, whether buried as green manure (Pikerton et al., 2000;Larkin & Griffin, 2008) or whether in the form of by-products of oil extraction from seeds (Lazzeri, et al., 2004;Palmieri, 2005;Sachi et al., 2005).The biofumigation effect of organic amendments on pathogens is due to, among other factors, toxic vola- tile compounds, anaerobic conditions and the increasing suppressive capacity of the soil (Tenuta et al., 2002;Everts et al., 2006;Bonanomi et al., 2007;Katase et al., 2009;Arriaga et al., 2011;Oka, 2010;Núñez-Zofio et al., 2011).The glucosinolate sinigrin of seed meal B. carinata pellets showed activity against Meloidogyne nematodes (Lazzeri et al., 2010), but not all Brassicaceus pellets are effective to the nematode control, which must be taken into account when selecting the amendment to the management of Meloidogyne (Zasada et al., 2009).The hydrothermal effect of solarization (Katan, 1981) has a direct effect on pathogens (Gamliel et al., 2004;Katan, 2005;Wang et al., 2006) and an indirect effect since it speeds up the decomposition of the organic amendment applied.Combining soil solarization with sorghum green manure reduced the nematodes populations more that the untarped sorghum but the combination of solarisation with broccoli residues did not reduce nematodes populations compared with untarped broccoli (Zasada et al., 2010).
In our case, the volatile compounds were not measured during the BS process but they may have been involved in reducing the incidence of M. incognita when compared the reduction obtained in October with BP and BP + M in second and third years.However, no such effect was observed in the second and third years with BP + M since the GI in October was similar to that of the control.Neither was nematode control improved when BP were mixed with M on both dates.An additive or synergic effect might have been expected between both amendments since M provided good control of M. incognita in pepper (Guerrero et al., 2007;Ros et al., 2008) as did the pellets alone (Guerrero et al., 2009).However, the interaction seems to be negative.In our conditions and soils with a pH > 8.0, decomposition of the amendments generates NH 3 , whose effect on fungi and nematodes, as described by Riegel & Noe (2000) and Everts et al. (2006), would explain the extent to which these pathogens are controlled by BS in similar conditions.The organic carbon may negatively influence the toxicity of glucosinolate-hydrolysis products (Morra, 2004).It seems that the organic matters of fresh sheep manure are not suitable to address the complex relationships than occur when using plant tissues as a source for pesticidal compounds.
It seems that the intrinsic water content of Brassicaceus tissues is insufficient to allow maximum hydrolysis of glucosinolates to isothiocyanates (Matthiessen, 2004).The addition of water performs the dual func-tions of boosting this process and of serving as a carrier for the isothiocyanates so formed into the soil.In our case the water absorption by fresh manure (M) could reduce the available water below the needs for hydrolysis of glucosinolates in pellets of B. carinata (BP).This might explain the lower efficiency of BP + M treatment compared to BP treatment in reducing the incidence of Meloidogyne.
Differences were observed in the soil oxygen content between the treatments (Guerrero, data not shown).The accumulated number of hours with oxygen levels below 5% or 1% was greater in BP + M than in the BP treatments and greater in August than in October for the same treatment.In the BP treatments the effect on the decrease of oxygen content disappeared after 3-4 weeks, while in BP + M it remained until the end of the solarization process.Anaerobiosis mechanisms may explain the differences in the incidence of M. incognita observed between the treatments and the control but not between BP and BP + M. Núñez-Zofío et al. (2011) observed differences between B. carinata pellets (BP) + Sinapis alba green manure and fresh manure (M) BS treatments for the control of Phytophthora sp. in pepper, but in the opposite direction as we observed for M. incognita.Núñez-Zofío et al. (2011) and Núñez-Zofío (2012) found greater numbers of bacteria, actinomycetes and Pseudomonas spp. in soils containing pepper crops biodisinfected with B. carinata pellets (BP) + Sinapis alba green manure than in soils disinfected with M or semi-composted sheep manure and the control, finding a positive correlation between total microbiota and the suppressive capacity of the soil after BS accompanied by a similar incidence of Phyptophthora spp. in the crop.We did not measure the microbiota but the effects on the incidence of the pathogen (M.incognita) differed between BP and BP + M the third year, which suggests that the possible effect in soil suppressiveness was not the main factor acting in the BS action mechanism.
Soil temperature seems to have been the main component of the effect of BS on M. incognita.In the August treatments of the third year more than 150 accumulated hours of temperatures above 42°C were recorded in BP at 15 cm depth and in BP + M at 15 and 30 cm; at this last depth the figure was 12.5 h for BP.Wang & McSorley (2008) found the accumulated exposure of more than 15 h to temperatures of 42°C were lethal for the eggs and juveniles 2 of M. incognita.In our case, this lethal exposure time was obtained for all the treatments and at both soil depths in the third year, at 15 cm the second year and at both depths the first year.However, the GI differed between treatments the second and third year.The time with temperatures above 38°C exceeded 400 h the second year at 30 cm in BP and at 15 and 30 cm in BP + M and only at 15 cm in BP the third year.Although Wang & McSorley (2008) suggested that 390 h at 38°C were necessary to kill all J2, this would still not explain the differences in GI between the treatments.Exposure of J2 and eggs to sublethal periods (< 13 h) of temperatures above 42.5°C may have an accumulative effect (Heald & Robinson, 1987), while temperatures that vary between 22°C and 42°C considerably reduce M. incognita J2 populations and eggs (Wang & McSorley, 2008).However, a reduction in the populations as a result of solarization is not always reflected in the adequate control of the damage caused to crops, which, according to Wang & McSorley (2008) would be related to low temperatures at greater depths of the soil.Populations remaining in the deep soil would recolonized and infest the damaged roots (Hewlet & Dickson, 1991), as occurred in the untreated control of long term crops (Chellemi et al., 1993).In our case, the 7-month crop cycle would have facilitated the multiplication of M. incognita and reinfestation of the plants, particularly in the autumn treatments, when the temperature never exceeded 38°C at either depth in any of the years.
Despite the fact that solarization only has a short term impact on nematode populations (Wang & McSorley, 2008) such an effect can be improved in conjunction with organic amendments, both in warmer and cooler periods of the year.The action of the volatile compounds released during the decomposition of BS amendments, the possible increase in the microbial community, and the concomitant suppressive effect of the soil and the reduction of the soil oxygen content, together, would explain the damage caused to nematodes by the August treatment.This reduced incidence of M. incognita enabled yields of pepper fruit similar to those obtained by MB.While in Autumn BS of greenhouse soils used for growing pepper in southeastern Spain cannot necessarily be recommended for the total control of M. incognita, such treatment can be used to alleviate the effect of soil fatigue due to microbiological causes (Martínez et al., 2011).The use of cultivars or plants grafted on rootstock that carry genes resistant to M. incognita may be considered as a complement to BS for the control of soil pathogens (Guerrero et al., 2012).

Table 1 .
Incidence of Meloidogyne incognita (galling index, GI and % of galled plants)

Table 3 .
Percentage of hours accumulated for each range of soil temperatures at 15 and 30 cm depth in each treatment for 27 days in August and 40 days in October (first season), for 30 days in August and 41 days in October (second season) and for 44 days in August and 41 days in October (third season)

October August October August October August October August October
BP + M: biodisinfestation with Brassica carinata pellets + fresh sheep manure; BP: biodisinfestation with Brassica carinata pellets.