Phytophthora capsici is one of the main soil-borne pathogens that causes root and crown rot of protected pepper ( Capsicum annuum L.) crops worldwide ( Ristaino & Johnston, 1999 ) and in the Basque Country (northern Spain) ( Riga et al ., 2000 ; Larregla et al ., 2015 ), an area characterized by a humid temperate climate. This pathogen species produces survival spores (oospores) that are capable to persist in the soil and act as the main initial inoculum that causes prima­ry infec­tions in the next crop cycle ( Erwin & Ribeiro, 1996 ). The presence of P. capsici is also reported in the southeast of Spain with a warm dried climate causing root and crown rot disease, not only in solanaceous plants (pepper, eggplant and tomato) but also in curcubitaceous like zucchini, watermelon and cucum­ber ( De-Cara-García et al ., 2018 ). Optimal, minimum and maximum tem­peratures for mycelia growth of P. capsici have been reported in the range of 25-32°C, 5-7°C and 38-42°C ( Erwin & Ribeiro, 1996 ). Oospores survival was scarcely affected at constant tempera­tu­res ≤ 35°C in controlled laboratory conditions in moistened soil. Required exposure times for thermal inactivation of P. capsici oospores were 199-22-6.6-4.7-1.0 h at 40-45-47.5-50-53°C of constant temperatures, respectively, in these constant temperature regimes. Regarding the cycling temperature regimes, 4 h-40°C regime killed 100% of oospores after 28 days, while the 5 h-35°C regime after 70 days killed only 75% ( Etxeberria et al ., 2011b ).

In greenhouses of the Basque Country, pre-plant soil disinfestation with chemical fumigants has been traditionally used to control pepper root and crown rot, but social changes and legal limitations on the use of chemical disinfectants boosted the evaluation of environmentally friendly and healthy alternatives of soil-borne pathogen control in the last years ( Colla et al ., 2012 ). One of these alternatives was the implementation of fresh green manure into the soil, which was reported to have a significant effect on soil-borne pathogen diseases ( Larkin, 2013 ; Lacasa et al ., 2015 ).

In some cases, the pathogen can be established in soils but the disease becomes progressively less severe even though the pathogen persists in soil, which is known as suppressiveness ( Cook & Baker, 1983 ; Chandrashekara et al ., 2014 ). The suppression of soilborne pests by naturally occurring biocides released in soil when tissues of Brassicaceous plants or fresh green manure crops are decompose in soil is known as biofumigation ( Kirkegaard et al ., 1993 ; Angus et al ., 1994 ). Biofumigation using Brassicas residues as fresh green manure ( Angus et al ., 1994 ) and as rotation crops fitted into farming systems ( Rudolph et al ., 2015 ), is an effective practice for the control of soil-borne pathogens while responding to environmental and health concerns as well as legal limitations ( OJ, 2009 ) of soil chemical disinfestation. Among Brassicas, Sina­pis alba (white mustard) was reported to be effective in soil-borne pathogen control in different crops like root rot in common Mediterranean crops ( De-Cara-García et al ., 2018 ), root and crown rot in pepper and tomato ( Morales-Rodriguez et al ., 2014 ) and pow­de­ry scab in potato crops ( Larkin, 2013 ).

Under certain conditions, soils can develop a suppressive effect which means that pathogens hardly establish in these soils or develop disease in the crop ( Shurtleff & Averre, 1997 ). The main mechanisms that explain the suppressive effects of the use of Brassica species for soil-borne pest control are the biocidal compounds released into the soil when Brassica tissues are decomposed ( Kirkegaard & Sarwar, 1998 ; Sarwar & Kirkegaard, 1998 ; Bending & Lincoln, 1999 ; Manici et al ., 2000 ; Matthiessen & Kirkegaard, 2006; ) and the increased populations of antagonistic organisms after the incorporation of organic matter in the soil ( Cohen et al ., 2005 ; Mazzola et al ., 2001 ).

The effectiveness of biofumigation combined with solarization, solar heating by covering a soil with a transparent plastic film ( Katan & Gamliel, 2014 ), has been described in Mediterranean areas with soil-borne pathogens problems ( Lacasa et al ., 2015 ; De-Cara-García et al ., 2018 ). In the case of areas with humid temperate climate, in which temperatures are insufficient for direct or indirect effects on pathogen control, the application of biosolarization in winter could contribute to control soil-borne pathogen. In this case in which temperature would probably not be high enough to produce solarization effects, it is more proper to use the term biodisinfestation.

In the Basque Country, pepper crops are grown in un­heated greenhouses from early-spring to early-autumn. Thus, the proper time to practice biodisinfestation in this area can be before (winter season) or after (fall season) the period in which the greenhouse is occupied by the pepper crop cycle ( Arriaga et al ., 2011 ). However, biodisinfestation at the end of winter can be considered a more viable option than in autumn, because of its greater proximity in time with the crop cycle, the progressive increase of solar radiation and consequently in temperature and microbial activity in the plastic-sealed amended soil. Brassica green manures can influence soil suppressiveness by several mechanisms different to the glucosinolates and isothio­cyanates pathway, such as indirect effects on the pa­tho­gen associated with changes in the populations of antagonistic organisms, as well as the more direct suppressive effects of compounds released from the tissues that may not be related to glucosinolates or isothiocyanates. In addition, other mechanisms inclu­de beneficial impacts on organic matter, nutrition, soil structure and erosion control which are common to most green manures ( Mcguire, 2003 ; Matthiessen & Kirkegaard, 2006 ; Prasad et al ., 2015 ). Thus, the establishment of the crop after an autumn-winter cover crop of Brassica regardless of its glucosinolate content, could be regarded as an adequate control method for Phytophthora root and crown rot.

The objective of this study was to evaluate the effectiveness of winter biodisinfestation as green manure with an autumn-winter cover crop cycle of S. alba (white mustard) on the survival of P. capsici oospores in the agroclimatic greenhouse conditions of the Basque Country, a humid temperate area with lower temperatures than the Mediterranean region.

Soil temperature remained below certain levels (40-53°C) that are considered critical to the survival of P. capsici oospores ( Etxeberria et al ., 2011b ). An absence of thermal inactivation was observed in this greenhouse experiment, located in the humid temperate Atlantic agroclimatic conditions of northern Spain. The low soil temperatures registered in our experiment (averages of 13.5 and 14.7°C at 15 and 30 cm depth respectively) would explain the limited production of biocidal compounds by decomposition from Brassica tissues and the reduced efficacy against P. capsici bu­ried inoculum, in line with the observations of Gamliel & Stapleton (1993) , Wang et al . (2009) and Prasad et al . (2015) in other pathosystems. These authors observed that some biocidal compounds, such as methyl sulfide and dimethyl disulphide, were only detected in heated Brassica amended soils and not in the unheated amended soils.

Our results are in line with the experiments performed by Lacasa et al . (2015) in paprika crops in open field conditions in Western Spain (Extremadura), in which biodisinfestation in spring with S. alba fresh green manure at low soil temperatures (below 25 °C for more than 80% of exposure time) did not improve infectivity reduction of Phytophthora nicotianae chlamydospores introduced inoculum. Similar results were obtained by Rodriguez-Molina et al . (2016) in the same region after spring biodisinfestation with defatted seed meal Brassica pellets. In contrast, these authors observed that spring biofumigation with plastic cover with Brassica pellets plus S. alba fresh green manure had no significant effect on the survival of P. nicotianae but caused reduction in chlamydospores infectivity.

Conversely, survival of P. capsici oospores in Mediterranean agroclimatic conditions was much reduced or almost extinct when soil temperatures were high during the biodisinfestation treatment. This was the case of greenhouses located in southeastern Spain that practice biosolarization in August-September with exposure times of six weeks and that apply fresh sheep manure mixed with other types of organic amendments such as fresh green manure of several Brassicaceae ( Lacasa et al ., 2015 ), defatted Brassica carinata seed meal dehydrated pellets ( Guerrero et al ., 2010 ) or several agro-industrial byproducts such as sugar-beet molasses ( Lacasa et al ., 2010 ) or Brewers' spent grain ( Guerrero et al ., 2014 ). In all these studies, depending on amendment treatments, the amended and plastic-tarped soil at 15 cm depth accumulated between 370 and 545 h at temperatures above 40°C. These high temperatures enhance the effectiveness of the treatment ( Katan & Gamliel, 2014 ) and the release of a greater variety of biocidal compounds ( Matthiessen & Kirkegaard, 2006 ; Prasad et al ., 2015 , 2016 ). Thus, a synergic effect between temperature and the release of Brassica biocidal compounds might account for the greater survival reduction observed in Phytophthora inoculum.

However, the oospores infectivity results (Table 2) suggest that the application of S. alba as a green manure can increase the suppressive capacity of the soil since disease development was significantly delayed in the biodisinfestated treatments in both years when compared to the non-treated control treatments at both soil depths. This suppressiveness seems to be mediated by the increase in the soil's microbial activity linked to the organic matter decomposition of fresh green manure and changes in the soil microbioma composition which are involved in the suppression of several soil-borne pathogens ( Larkin et al ., 2007 ; Mazzola et al ., 2017 ). The infectivity results of our experiment could be partially explained by the same mechanism observed by Mazzola et al . (2001) in the suppression of R. solani apple root rot by proliferation of soil microbes after Brassica napus seed meal amendment regardless of its glucosinolate content.

The low temperatures recorded in our experiment could explain the low reduction of P. capsici oospore survival and suggest that biofumigation mecha­­nism pathway glucosinolates-myrosinase-isothiocyanates has had little effect on the low suppressiveness expressed in our infectivity bioassay. The generation of toxic compounds (mainly isothiocyanates) from the glucosinolates present in the species of Brassicas can inhibit the germination of oospores, as demonstrated by Manici et al . (2000) for oospores of Pythium spp., preventing or delaying the establishment of disease. On the other hand, the culture and subsequent biodisinfestation with S. alba significantly increased the dehydrogenase and glucosidase activities with respect to the non-treated control (percentages increases of 66% and 17%) in the first year (2009) and the dehydrogenase, glucosidase, urease and phosphatase activities (percentages increases of 90%, 31%, 52% and 37%) in the second year of reiteration (2010). The increase of microbial activity can influence the control of soil pathogens through antagonistic mechanisms such as parasitism, competition and antibiosis ( Janvier et al ., 2007 ), which could be linked to the initial observed delay in oospores infectivity in the biodisinfestation treatment with S. alba (Fig. 1; Table 2) and in agreement with other studies conclusions ( Mazzola et al ., 2001 , 2018 ; Kirkegaard & Matthiessen, 2004 ; Cohen et al ., 2005 ; Núñez-Zofío et al ., 2012 ; Hewavitharana & Mazzola, 2016 ).

Suppressive effects of P. capsici were observed on the subsequent greenhouse pepper crop after applying a fresh green manure of S. alba as opposed to the utilization of B. carinata pellets which did not reduce the disease in previous experiments with greenhouse pepper crops in northern Spain ( Núñez-Zofío et al ., 2011a , b ). The rhizosphere effect derived from the presence of S. alba plants during winter might be responsible for the observed higher soil biodiversity, possibly resulting in higher suppressiveness and lower disease incidence in the subsequent pepper crop cycles grown in years 2009 (2.8% in S. alba + plastic vs 42.6% in non-treated control) and 2010 (14.0% in S. alba + plastic vs 29.6% in non-treated control) ( Núñez-Zofío et al ., 2011b , 2012 ). This could be more related with the long-term (several weeks) suppressive effects of the use of Brassica linked to mechanisms of increase of microbial activity ( Mazzola et al ., 2001 ; Cohen et al ., 2005 ) than with the short-term effect (a few days) linked to mechanisms of generation of biocidal isothiocyanates derived from glucosinolates hydrolysis through the myrosinase enzymatic system ( Kirkegaard & Sarwar, 1998 ; Sarwar & Kirkegaard, 1998 ; Matthiessen & Kirkegaard, 2006 ; Prasad et al ., 2015 , 2016 ).

According to Lacasa et al . (2015) , in the Mediterra­nean climate conditions of Southeastern Spain, the ther­mal effect of summer biosolarization had more impact than the biofumigant effect of compounds generated by Brassicas on P. capsici oospores viability. In contrast, in winter biodisinfestation with Brassicas in the humid temperate climate of northern Spain, thermal and biofumigant effects were insufficient for P. capsici oospores short-term inactivation, in agreement with reduced efficacy of soil solarization by cabbage amendment in climates such as in the South-eastern United States, where cloud cover and rainfall interrupted the heating process of the soil to eliminate P. capsici oospores at a depth of 25 cm ( Coelho et al ., 1999 ). However, long-term suppressive effects were achieved and final crop disease incidence was reduced. Reiteration of S. alba crops as fresh green manure could contribute to achieve long-term suppressive effects with the increase in soil's microbial activity and mobilization of specific antagonistic soil bacterial populations that reduced crop disease incidence in the subsequent pepper crop cycles ( Mazzola et al ., 2001 ; Cohen et al ., 2005 ; Núñez-Zofío et al ., 2011b , 2012 ).

In view of the obtained results, it can be concluded that winter biodisinfestation with fresh Brassica green manure is a promising management strategy for Phytophthora root rot control of protected peppers crops in regions of humid temperate climate such us northern Spain. Nevertheless, further research will be necessary to guarantee an effective Phytophthora biodisinfestation by fitting green manure species in combination with other fresh organic amendments.

The authors thank Berdaitz Juaristi, Javier Elorrieta and Leire Abaunza for their helpful support to carry out greenhouse experimental field trials in NEIKER.