Introduction
Greenhouses of pepper (Capsicum annuum L.) in southeast Spain suffer from serious economic losses caused by the oomycetes Phytophthora capsici and P. nicotianae, which contaminate most of the region's soils (Lacasa et al., 2013). Both of these oomycetes limit the crop, leading to important losses in plants and harvests if preventive measures are not adopted before planting (Lacasa et al., 2010; Guerrero et al., 2013). Nowadays, P. nicotianae is the most prevalent (Lacasa et al., 2013; Blaya et al., 2014), favored by the climatic conditions in the greenhouses and farming practices. Chlamydospores and/or oospores may survive for up to 6 years (Erwin and Ribeiro, 1996). The main soil-borne pathogens of protected peppers are Phytophthora spp. and Meloidogyne incognita in addition to soil fatigue issues which are specific of this crop. The previous soil phytopathological problems are both caused by the repeated growing of pepper crops in the same soil for over 30 years (Guerrero et al., 2014).
Between 1982 and 2005, soil disinfestation with methyl bromide was the way to palliate the effects of Phytophthora root rot in more than 95% of the greenhouses (Lacasa et al., 2010). The mix of chloropicrin and 1,3-dichloropropene has replaced methyl bromide in over 70% of the surface area. Biodisinfection is used in 20% of the surface area, combining solarization with biofumigation (Lacasa et al., 2010; Guerrero et al., 2013).
The combination of solarization with biofumigation shows that the two methods have synergic effects (Katan, 2005). Sealing the soil with plastic produces the gas retention that occurs during organic matter decomposition (Bonanomi et al., 2007; Oka, 2010). If saturation levels are reached when the soil is wetted, then the effects of anaerobiosis are also added and phytoparasites and pathogens are affected (Blok et al., 2000).
If water saturation levels are not reached, but organic amendments containing a high carbon content are used anaerobiosis occurs under the plastic during biodecomposition (anaerobic soil disinfestation, ASD) which affects the pathogens (Shennan et al., 2014; Rosskopf et al., 2015). During the degradation process of the organic matter, changes occur in the composition of the microbiota that confer a suppressive character against some pathogens (Mazzola, 2011; Núñez-Zofío et al., 2011; Mazzola and Manici, 2012; Mazzola et al., 2012). The suppressive effects and the anaerobiosis depend on the soil characteristics, the organic amendment used, the soil temperature, the characteristics of the microbiota and the disinfectant effectiveness or the pathogens susceptibility (Butler et al., 2012, 2014). The choice of the organic matter used in the biodisinfection is one of the determining elements for its efficacy. Bonanomi et al. (2007) suggested that the application of specific organic matters: crop residues and organic wastes, compost and peat could be useful to increase soil suppressiveness. Núñez-Zofio et al. (2013), found an effective biofumigant in beetroot stillage. Guerrero et al. (2013), checked the effect of Brassica carinata pellets on Meloidogyne incognita in pepper greenhouses in southeast Spain.
European Union restrictions on the use of chemical products, as well as the increase in the surface area dedicated to ecological agriculture, require the use of soil organic amendments for soilborne disease management. Biodisinfection using fresh sheep manure in August gives good results in controlling Phytophthora in greenhouses in southeast Spain, although this is not the case when the process is performed after the start of September (Guerrero et al., 2010). The majority of the greenhouses soils in the region are clay loam (Haplocalcids), basic pH, with an organic matter content between 1.5 and 2.5%. The climate is Mediterranean with marine influence, hot dry summers, and mild winters. The greenhouse pepper crop is planted in December-January, and finishes in August-September.
In order to fit the start of the biodisinfection to the end of the cropping season it is necessary to commence in October. Using the amendments that are typically used in August (fresh sheep manure), the biodisinfection does not reduce the levels of pathogens, and are thus unviable (Guerrero et al., 2010, 2013; Lacasa et al., 2010), so it is necessary to improve the biodisinfection effects by adding amendments that strengthen the anaerobic effect or increase soil temperature, both effects by the organic matter decomposition.
Moreover, the southeast of Spain is in a vulnerable zone due to nitrates (Directive 91/676/CEE, of 12 December, relating to the protection of waters against contamination produced by agricultural origin nitrates) which means that there are restrictions on the contribution of nitrogen. This premise severely limits the use of organic matter in the zone.
The objective of this study was to obtain biodisinfection amendments that are effective in controlling Phythophthora in pepper greenhouses with low temperatures whilst also reducing the amount of fresh organic amendments.
Discussion
In our trial, the temperatures obtained during the biodisinfection carried out in October with the amendments of wheat husk+fresh sheep manure, sunflower pellets, and fresh sheep manure exceeded 38°C in the first season, varying between 51 and 57 h, but no in the second. At both seasons soil temperatures accumulated in October during the biodisinfection in the greenhouse were considerably lower than those obtained for the month of August in the same zone (Guerrero et al., 2019). Etxeberria et al. (2011) found that for 1,680 h at 15–35°C, the survival of the oospores ranged between 88 and 36%. The oospore viability in the two seasons was reduced by the presence of minimum temperatures above those reported by Etxeberria et al. (2011). The results also concur with those obtained by Lacasa et al. (2015) in which biodisinfection in spring with Sinapis alba fresh green manure did reduce the infectivity of Phytophthora nicotianae chlamydospores introduced inoculum (Gandariasbeitia et al., 2019). The infectivity of the surviving inoculum corresponded to an insufficient reduction of the inoculum buried in the bags. Coelho et al. (2000) considered that inoculum levels in excess of 1 propagule per gram of soil are necessary in order to detect the surviving inoculum using plant indicators in bioassays.
The biodisinfection with the assayed amendments induce anoxia in the soil for several weeks, which is one of the factors indicated for Gamliel et al. (2000) as cause of the decrease in the pathogen populations and of the efficacy of the disinfestation. In the first season, the level of oxygen in the soil was below 2% for more than 64% of the disinfestation time for S and FSM treatments, and over 35% of the time in WH + FSM. In the second season, the corresponding time was more than 57% in FSM and over 84% in S and for WH + FMS. The production of sporangia and the mycelial growth of several species of Phytophthora (including P. capsici) was considerably lower when the oxygen level was at 1% of oxygen in the crop solution. Below 2.5% of oxygen, the formation of sporangia is reduced and also the mycelial development of P. cinnamomi, affecting the viability of the inoculum (Davison and Tay, 1987).
The efficacy of the biodisinfection when temperatures are low improves with the contribution of amendments that are rich in organic matter or by increasing the amount of carbon that is applied to the soil. The contribution of 4 mg C g−1 of soil has been recommended to improve the efficacy of ASD at low temperatures (Butler et al., 2014; Rosskopf et al., 2015). It is recommended that the carbon of the organic amendment should be labile, so that it can easily and rapidly be degraded by the soil microorganisms and create conditions for persistent anaerobiosis. Additionally, an adequate ratio of C/N must be considered so that, on the one hand, the N that is applied is enough so that N is not immobilized by the soil microorganisms in the degradation/mineralization of the organic amendments and, at the same time, there is not a potentially leachable excess of N. In our trials, the amendments presented a balanced composition, and ratios of C/N within the limits that are considered as adequate for biodisinfection and the generation of hypoxia.
The efficacy or failure of soil biodisinfection may be due to the soil characteristics, the degree of moistening, the electrical conductivity, the characteristics of the plastic, and the period of solarization (Chellemi, 2002). The temperate-humid climate has obstructed the application of biosolarization, very extended in Mediterranean horticultural areas such as South-eastern Spain. However, previous experimentation with different amendments, doses and application times compatible with crop cycles showed the applicability of these practices in the humid temperate climate region of Northern Spain (Ojinaga et al., 2020).
In the north of Spain, the survival of oospores of P. capsici was reduced at 15 and 30 cm when the biodisinfection was applied in August with both fresh and semi-composted manure (Núñez-Zofío et al., 2012). Using rice bran, rapeseed cake, or grape pomace as the carbon source, Serrano-Pérez et al. (2017) obtained significant reductions in chlamydospores viability and inoculum infectivity of pepper plants by P. nicotianae, when biodisinfection was carried out in low temperature open air conditions of the spring season in south western Spain.
Despite the low disinfectant efficacy assessed by the reduction in the survival of P. capsici oospores in sub-optimal conditions for solarization (absence of thermal inactivation), improvements were observed in the suppressiveness and in the chemical, physical and biological soil properties when the organic amendments were applied in biodisinfection, which caused a reduction of crop diseases and a yield increase (Núñez-Zofío et al., 2012; Rosskopf et al., 2015; Gandariasbeitia et al., 2019). It was concluded that repeated biodisinfection for the control of Phytophthora root and crown rot in protected pepper crops located in temperate climate regions can improve soil quality and suppressiveness, as well as enabling reduced doses of the organic amendment needed for biodisinfection. However, this disinfestant efficacy worsened when the biodisinfection was performed in March (Arriaga et al., 2011), and also when it was carried out in September (Núñez-Zofío et al., 2011).
One strategy employed when biodisinfection fails due to the climate or crop cycles is to turn to anaerobic disinfection. This strategy has been applied in limiting solarization conditions in Japan (Shinmura, 2000; Momma et al., 2005, 2006, 2010, 2011; Katase et al., 2009) and also in the Netherlands (Blok et al., 2000; Agtmaal et al., 2015).
The effect of the application of organic matter produced a yield increase in both seasons. Although there was no naturally occurring presence of pathogens, the increased yield in the biodisinfested plots might be explained by the reduction of soil fatigue in the crop. The fatigue accumulated in greenhouses used for pepper monocropping is highly specific toward pepper (Guerrero et al., 2014). The greenhouse assayed in this study fits the conditions of pepper monocropping. Biodisinfection with fresh sheep manure of greenhouses used to grow pepper crops, improved the physical and chemical characteristics of the soil (Fernández et al., 2005). The application of wood waste and sunflower husk biochar improved soil porosity and the structural stability of soil aggregates (Sokołowska et al., 2020). Improvement of soil water infiltration, soil drainage, and roots aeration have been associated with a better control of crop root rot caused by soilborne pathogenic species of Phytophthora (Ristaino, 1991).
The principal cause of this fatigue is considered to be biotic and related with the extent to which the soils are contaminated with Fusarium sp. (Martínez et al., 2009, 2011). It is additionally known that the fatigue accumulated in greenhouses used for pepper monocropping is highly specific toward pepper (Guerrero et al., 2014). In soils contaminated by Fusarium spp. and Phytophthora spp., Song et al. (2020a,b) obtained a significant reduction in the incidence of Fusarium and Phytophthora in strawberry crops in conditions of biodisinfection with chicken manure or with maltose as carbon sources at 28°C and covering the soil with total impermeable film. They also observed a yield improvement related with the increase of soil available nutrients after the biodisinfection treatment.
In those plots where FSM was not used in this study, the yields were similar to those in which it had been used. This fact is of great interest since the cropping zone of Southeast Spain is subject to organic matter restrictions. Moreover, it is known that the incorporation of organic correctives improves soil properties and fertility, as reported by Bonanomi et al. (2007).
The results obtained with the carbon-rich and balanced amendments and in time periods that are compatible with the pepper crop cycle in greenhouses in southeast Spain, showed a yield improvement. This increased yield may also be due to the effect highlighted by Núñez-Zofío et al. (2011) that repeated biodisinfection treatments in temperate climate areas improve soil suppressiveness and quality. This would enable growers to extend the crop cycle and thereby the corresponding economic benefit.
In conclusion, using the specific organic amendments, significant reductions were obtained in the viability of the inoculum resting structures of P. capsici as well as increases in the marketable yield, when biodisinfection is performed on dates that are compatible with the pepper growing cycle in the Campo de Cartagena. This way of soil disinfestation is recommendable in the context of the strategies of sustainable integrated production.