Efficacy of Brassica carinata pellets to inhibit mycelial growth and chlamydospores germination of Phytophthora nicotianae at different temperature regimes

Scientia Horticulturae 216 (2017) 126–133

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Efficacy of Brassica carinata pellets to inhibit mycelial growth and chlamydospores germination of Phytophthora nicotianae at different temperature regimes Paula Serrano-Pérez ∗ , Carolina Palo, María del Carmen Rodríguez-Molina ∗ CICYTEX, Instituto de Investigaciones Agrarias Finca La Orden-Valdesequera, A-5 km 372, 06187,Guadajira, Badajoz, Spain

a r t i c l e

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Article history: Received 5 July 2016 Received in revised form 28 December 2016 Accepted 4 January 2017

Keywords: Biofence Biofumigation Brassica amendments Isothiocyanates Soilborne pathogens Paprika pepper

a b s t r a c t Phytophthora nicotianae causes root and crown rot disease in open field paprika pepper crops of Extremadura (western Spain). Sensitivity of the vegetative structures (mycelia and chlamydospores) of P. nicotianae to Brassica carinata pellets (Biofence) was evaluated in vitro at different doses (3, 6, 12 and 24 mg of pellet per plate) and temperatures (15, 20, 25 and 30 ◦ C). The effectiveness of the pellets varied depending on the dose. An inhibition effect that decreased with time, was observed in the mycelial growth and chlamydospores germination in plate. The highest dose of pellets tested (24 mg) was fungitoxic to mycelium regardless of temperature for all the isolates. Moreover, biofumigation was effective to suppres chlamydospores germination when the pellets were incorporated into the soil (1.5 and 3 g L−1 of soil) under different temperature regimes. In bioassays with pepper plants, both rates of B. carinata pellets (1.5 and 3 g L−1 of soil) reduced populations of P. nicotianae below the limits of detection of our bioassay (<2 CFU g−1 of soil) after a 4-week biofumigation treatment and totally controlled the disease. The results suggest that chlamydospores of P. nicotianae in the soil might be suppressed by the commercial rates of B. carinata pellets, although further studies are required in more real soil conditions to form part of integrated pest management program. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Phytophthora nicotianae Breda de Haan (=P. parasitica Dastur) is one of the most widespread and destructive soilborne plant pathogens (Erwin and Ribeiro, 1996) and it is responsible for heavy losses on a particularly high number of host plants (Panabières et al., 2016). In Extremadura region (western Spain), this pathogen is the principal causal agent of root and crown rot in open field paprika pepper crops, where the pathogen survives mainly as chlamydospores (Rodríguez-Molina et al., 2010). Currently, many of the compounds used in its control, such as methyl bromide, 1,3-dichloropropene, or chloropicrin, are not included in Annex I (Directive 91/414/EEC) and can only be used under special authorisation of the European Commission. As a result of restrictions on the use of chemicals, soil disinfestation with biofumigants has become a usual practice to control soilborne diseases. Biofumigation was originally defined as the sup-

∗ Corresponding authors. E-mail addresses: [email protected] (P. Serrano-Pérez), [email protected] (M.d.C. Rodríguez-Molina). http://dx.doi.org/10.1016/j.scienta.2017.01.002 0304-4238/© 2017 Elsevier B.V. All rights reserved.

pression of soil pest and diseases by biocidal volatile compounds, principally isothiocyanates (ITCs), released by species of Brassicaceae when glucosinolates (GSLs) in their tissues are hydrolysed by the myrosinase enzyme (Angus et al., 1994; Kirkegaard et al., 1993). This technique can be achieved by incorporating fresh plant material (green manure), seed meals (co-products from biodiesel production) or dried plant material treated to preserve ITC activity (De Corato et al., 2015; Gimsing and Kirkegaard, 2009). The use of fresh Brassicaceae material presents several negative aspects, such as the costly cultivation operations required, the loss of at least one growing cycle with consequent loss of income (Lazzeri et al., 2008) and the variation in biomass production, depending on climatic and soil factors. Seed meals of these species of plants have several advantages over green manures, including their availability throughout the year and their low moisture content, which allows storage with a stable GSL profile (Mazzola and Zhao, 2010). Brassica carinata A. Braun is the most studied species that provides ideal formulations for biofumigation (Porras and Dubey, 2011). A formulation of B. carinata defatted seed meal prepared in the form of pellets has been patented (Lazzeri et al., 2008) and its ability to suppress several soilborne pathogens has been proved (Garibaldi

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et al., 2010; Gilardi et al., 2016; Guerrero-Diaz et al., 2013; Morales˜ Rodríguez et al., 2014; Lazzeri et al., 2008; Núnez-Zofío et al., 2011; Wei et al., 2016). Seed meal is deffated to increase the content of GSLs, and this formulation permits the exploitation of concentrations of GSLs substantially impossible to achieve with the green manure technique (Lazzeri et al., 2008). Sinigrin is the predominant glucosinolate in the B. carinata pellets and releases toxic allyl isothiocyanate (AITC) after being hydrated (Lazzeri et al., 2008; De Nicola et al., 2013). The antifungal activity of pure AITC has been previously demonstrated (Mari et al., 2008; Rahmanpour et al., 2009; Sarwar et al., 1998; Sotelo et al., 2015) and its effect increases with increasing concentrations (Kurt et al., 2011; Taylor et al., 2014). High percentages of the total AITC potentially achievable are released few hours after hydratation (De Nicola et al., 2013; Lazzeri, 2014). The temperature is an extremely important factor because it affects the volatility and concentration of the ITCs produced during the hydrolisys of GSLs (Price et al., 2005). However, information about the influence of temperature on the effect of B. carinata pellets is limited. Other compounds, in addition to products of glucosinolate degradation, may be biologically active and thus contribute to disease control (Gamliel and Stapleton, 1993; Wang et al., 2009). Differences in sensitivity of fungal species to pure ITCs have been reviewed, and fungicidal concentrations of ITCs for different species of fungi may differ by an order of magnitude (Brown and Morra, 1997; Fenwick et al., 1983; Rosa and Rodríguez, 1999). Variation in sensitivity to different ITCs has been reported not only within species but also within isolates of the same species (MoralesRodríguez et al., 2012, 2014; Smith and Kirkegaard, 2002). The objetives of this reseach were (i) to determine in vitro the sensitivity on vegetative structures of P. nicotianae to B. carinata pellets under different temperatures and exposition time, (ii) to evaluate in vitro the effect of pellets incorporated into the soil on chlamydospores under different temperatures, and (iii) to test the effectiveness of field doses of pellets against P. nicotianae on pepper plants.

2. Material and methods 2.1. Phytophthora nicotianae isolates and chlamydospores inoculum production Inhibition of mycelial growth rate was studied in 11 isolates of Phytophthora nicotianae from two hosts and two locations. Four isolates (P11, P13, P26, P21) from diseased pepper plants and three isolates (T1, T3, T7) from diseased tomato plants grown in Extremadura (western Spain) and their pathogenicity had been previously tested (Rodríguez-Molina et al., 2010). Four isolates (P44-M, P53-M, P57-M, 1134) from diseased pepper plants grown in Murcia (southeast of Spain) were provided by the IMIDA. Only one isolate (P13) was used for all other trials. Isolates were grown on clarified V8 juice agar at 25 ◦ C for 3–5 days before starting all the inhibition trials. Chlamydospores were produced using the procedure proposed by Rodríguez-Molina et al. (2016) that modifies the methodology described by Tsao (1971) and Mitchell and Kannwischer-Mitchell (1992). Briefly, the isolate (P13) was grown on clarified V8 juice broth at 25 ◦ C for 10 days in dark. Then, the V8 juice broth was removed and the mycelial mat was submerged in sterile distilled water at 18 ◦ C in the dark for a minimun of 7 days. Once the chlamydospores were formed, the mycelial mat was rinsed in sterile distilled water and blended for 7 min in a homogenizer (MICCRA D-1, ART-moderne Labortechnik, Germany) with 50 ml of sterile distilled water and then sonicated (HD 2070, Sonoplus, Bandelin, Germany). The resulting suspension was centrifugated

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(2 min, 1760 rpm) and the pellet was resuspended in sterile distilled water. The total number of chlamydospores in the suspension was estimated with the aid of a Neubauer hemocytometer and viability of chlamydospores was determined by staining with rose bengal solution (Tsao, 1971). 2.2. In vitro inhibition of mycelial growth rate and germination of chlamydospores There was assayed the effect of volatiles released by hydratation of defatted seed flour of B. carinata prepared in the form of pellets (BioFence; Triumph Italia SPA, Cerealetoscana Group) on the mycelial growth rate and on the germination of chlamydospores of P. nicotianae. The pellets were crumbled with a mortar and pestle before using. Four doses (3, 6, 12 and 24 mg of dry pellet per plate), selected according the results of previous assays (Morales-Rodríguez et al., 2014), were compared at four different temperatures (15, 20, 25 and 30 ◦ C). A dose zero was included as control. In the inhibition of mycelial growth tests, one mycelial plug (5 mm in diameter) from actively growing colony of the isolate was transferred in the center of a 9-cm Petri plate containing 18 ml of V8 juice agar. Plates were incubated for 24 h at 25 ◦ C before being exposed to the biofumigant. In the inhibition of chlamydospores germination tests, the concentration of chlamydospores suspension was adjusted to 100 chlamydospores/ml and a 500 ␮l aliquot was uniformly spread on NARPH medium (cornmeal agar amended with natamycin 5 mg L−1 , ampicillin 250 mg L−1 , rifampicin 10 mg L−1 , pentachloronitrobenzene [PCNB] 133 mg L−1 and hymexazol 50 mg L−1 ). According to the method reported by Lazzeri et al. (2008) for Fusarium culmorum and Morales-Rodríguez et al. (2014) for P. nicotianae, the plates inoculated with either mycelium plugs or chlamydospores suspension were inverted and the biofumigant material was placed inside on the covers of plates. Plates without biofumigant material were used as control (dose zero). Biofumigation was started by hydrating the pellet with distilled water (1:2, w:v). The plates were sealed with Parafilm to avoid loss of volatiles released and incubated inverted in the dark at four temperatures: 15, 20, 25 and 30 ◦ C. Four replicates were prepared per temperature and dose of biofumigant. To evaluate the efficacy of biofumigation, radial growth of the isolate was measured (mean of two perpendicular diameters) at 24 h, to exclude the initial growth lag phase, and thereafter at 3 and 6-days incubation period for each temperature. The results were expressed as mycelial growth rate (mm/day). In the plates where no mycelial growth occurred after 6 days of incubation, the plate covers with biofumigant were removed and replaced by a new one without biofumigant and incubated for one week at 25 ◦ C in the dark, in order to evaluate the fungistatic or fungitoxic effect of the volatiles released by the biofumigant. In the plates of chlamydospores germination tests, the number of colony-forming units (CFU) from chlamydospores suspension was counted daily while independent colonies were distinguishable (5–10 days, depending on the temperature of incubation). Accumulation of heat units, expressed in degree-days, was used to analyze the effect of the dose of treatment on the germination of chlamydospores depending on the temperature and time of incubation. According to Lutz et al. (1991), a threshold temperature of 12 ◦ C was selected and calculation of degree-days was determined by the following formula: (incubation temperature −12) × (number of days of incubation). Results of chlamydospores germination of P. nicotianae were expressed as mean ratios rather than actual numbers of CFU recovered. Mean ratios were calculated by dividing the CFU g−1 obtained after each treatment by the CFU g−1 obtained in the untreated control at the same incubation conditions.

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2.3. Inhibition of germination of chlamydospores in soil Soil used in the experiments was a sandy loam soil (pH 6.5; 0.55% organic matter) collected in a field at the Agricultural Research Institute Finca La Orden-Valdesequera (Extremadura, western Spain). Prior use, soil was sieved (2 mm sieve) and autoclaved 1 h at 120 ◦ C twice in two consecutive days. A closed controlled-temperature system was set up to evaluate the effectiveness of the B. carinata pellet when it was added into the soil at different regimes of temperature. In this system chlamydospores of P. nicotianae were exposed to (i) all the compounds released into the soil solution during the biofumigation process or only to (ii) the volatile compounds generated during the process. The controlled system consisted of hermetic 1-l glass containers (14 cm height and 10 cm diam) that were filled with 50 g of dry soil mixed with the crumbled pellet and inoculated with 10 ml of suspension of chlamydospores to obtain a final concentration of 500 chlamydospores per gram of dry soil. Small inoculum bags with agryl cloth containing 5 g of soil and 500 chlamydospores per gram of dry soil were hung in the headspace of the container, avoiding contact with the bottom of the container. The containers were hermetically sealed, placed inside a programmable incubator in a complete randomized design with four replicates per treatment and temperature and incubated for one week. Two rates of pellets were assayed: 3 t ha−1 (BF3 = commercial rate, according to the manufacter’s recommendations; equivalent to 64.5 mg of dry pellets per container or 1.5 g L−1 of soil) and 6 t ha−1 (BF6 = double commercial rate; equivalent to 129 mg of dry pellets per container or 3 g L−1 of soil). The conversion between weight and area units was based on the soil bulk density of 1.163 g cm−3 and soil incorporation of 20-cm depht. Three control treatments were included: (i) soil inoculated and without pellets, (ii) pellets rate BF3 without soil and (iii) pellets rate BF6 without soil. The treatments were compared at four different constant temperatures (15, 20, 25 and 30 ◦ C) and at three different fluctuating temperatures (T1, T2 and T3). The fluctuating temperature regimes were: T1 (14 ◦ C for 7.5 h/day; 16 ◦ C for 12 h/day; 19 ◦ C for 4.5 h/day), which was the prevailing in soil at 15-cm depth in spring in western Spain (Rodríguez-Molina et al., 2016); T2 (27 ◦ C for 4 h/day; 30 ◦ C for 12 h/day; 33 ◦ C for 8 h/day) emulated the conditions of solarization in summer with sugar beet vinasse in a greenhouse in southeast Spain at 15-cm depth (Lacasa et al., 2010), and T3 (30 ◦ C for 4 h/day; 33 ◦ C for 8 h/day; 37 ◦ C for 8 h/day; 40 ◦ C for 4 h/day) simulated similar conditions than T2 but using fresh sheep manure as organic amendment (Lacasa et al., 2010). At the end of the incubation period, the containers were opened and the soil from the bottom and from the inoculum bags was analyzed according to the method by Mitchell and KannwischerMitchell (1992) to estimate the number of chlamydospores of P. nicotianae surviving after the treatments. Briefly, 5 g of soil were diluted in 0.25% water-agar (1:10, w:v) and five 1-ml aliquots from this dilution were placed onto each of 5 Petri plates with 12 ml of NARPH medium, giving the detection threshold of 2 CFU g−1 of soil. After 48 h of incubation in the dark at 25 ◦ C, the soil water-agar overlay was removed by gently with tap water. Macroscopically visible colonies of P. nicotianae were counted and reported as number of CFU g−1 of dry soil. Results of chlamydospores germination were expressed as mean ratios calculated at each sample by dividing the CFU g−1 obtained after each treatment by the CFU g−1 obtained in the untreated control in the same incubation conditions.

trial was conducted in 10-l plastic containers (33 cm height and 20 cm wide) with a mixture of autoclaved soil (1 h at 120 ◦ C) and V8 juice-vermiculite inoculum of P. nicotianae (Hoitink et al., 1977; Sujkowski et al., 2000). This inoculum was prepared in 1-l bags containing 650 cm3 of vermiculite amended with 350 ml of clarified V8 broth and autoclaved for 1 h at 121 ◦ C. The bags were inoculated with 10 agar plugs (∼20 mm in diameter) removed from 7-day-old culture of P. nicotianae (P13 isolate) and incubated at 25 ◦ C for 4 weeks before soil infestation at a rate of soil:inoculum 1:0.75 (v:v). Initial inoculum density was 37 ± 10 CFU g−1 of soil. For uninfested control treatment (UC), the agar plugs were removed from V8 Petri plates without culture. Then, pellets were mixed with the soil at doses of 1.5 g L−1 (BF3) and 3 g L−1 of soil (BF6). Infested soil without pellets (C) was included as control treatment. There were four replicates for each treatment. Containers were irrigated to water-holding capacity (volumetric water content at 0.21 m3 m−3 ), sealed with plastic film (PE 0.05 mm) and kept in a growth chamber with a 16 h light at 19 ◦ C/8 h dark at 14 ◦ C cycle. Four weeks later, plastic covers were removed and survival of inoculum was determined by soil dilution plate method (Mitchell and Kannwischer-Mitchell, 1992) with 0.25% water-agar on NARPH medium, giving the detection threshold of 2 CFU g−1 of soil. Results were expressed as CFU g−1 of dry soil. Ten pots were filled with the mixture of each container and one pepper seedling (cv. Jaranda) at the 2–4-true-leaf stage was transplanted into each pot, with its roots placed in contact with the treated soil. Plants were grown in a greenhouse and disease symptoms (wilting and crown or root rot) were recorded every week over a period of two months. As plants died, fragments of roots and crown were plated on NARPH medium to reisolate the pathogen. At the end of the bioassay, the root systems of all plants were examined for the presence of disease symptoms and analyzed. Results of infectivity are expressed as the percentage of diseased plants. 2.5. Data analysis Data of mycelial growth rate were analysed with analysis of variance for repeated measures (rmANOVA) with Bonferroni’s correction for means comparisons, including statistical significance for measure time as within-subjects factor (3 and 6 days of treatment) and isolate, dose and temperature as between-subjects factors, as well as the interactions between them. Sphericity was not considered since within-subjects factor had only two levels. For chlamydospores germination, data were analyzed by multiple regression analysis and the model Y = a + b1 X1 + b2 X2 , in which Y = germination percentage, X1 = dose of pellet and X2 = degreedays of incubation. The method was stepwise and the model was expanded to include the interaction of dose and degree-days (b3 X1 X2 ) as a third variable. Data of germination of propagules in soil under different temperatures and survival and infectivity of inoculum of in vivo trial were analysed using nonparametric Mann-Whitney U test for pairwise comparisons with Bonferroni’s correction to adjust the probability. Unless otherwise specified, effects and differences were considered significant at P < 0.05. All analyses were performed with the software package SPSS version 20.0 (SPSS Inc., Chicago, Illinois, USA). 3. Results 3.1. In vitro inhibition of mycelial growth rate and germination of chlamydospores

2.4. Bioassay with pepper plants after soil biofumigation An experiment was carried out to evaluate the effectiveness of B. carinata pellets against P. nicotianae on pepper plants. The

Mycelial growth rates of the P. nicotianae isolates at different temperatures after 3 and 6 days of exposure to different doses of B. carinata pellets are presented in Table 1. The results of

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Table 1 Mycelial growth rate (mm/day) of the P. nicotianae isolates at different temperatures after 3 and 6 days of exposure to different doses of B. carinata pellets. Per time and dosez , mycelial growth rate (mm/day)y 3 days

6 days

Isolate

Temp.

0 mg

3 mg

6 mg

12 mg

24 mg

0 mg

3 mg

6 mg

12 mg

24 mg

P11

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

6a 9a 10a 10a

3b 7b 10a 9b

0c 7b 9b 6c

1c 1c 1c 0d

0c 0c 0c 0d

6a 9a 11a 11a

5b 8a 11a 12a

3c 9a 11a 13a

0d 9a 14a 9b

0d 0b 2c 0c

P13

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

7a 10a 12a 11a

3b 9a 9b 9b

1c 5b 7c 6c

1c 1c 2d 1d

0c 1c 0e 0d

7a 10a 12a 12a

5b 9a 11a 13a

3c 9a 12a 14a

0d 7c 12a 12a

0d 0d 0b 0b

P21

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

5a 7a 10a 9a

1b 4b 9b 6b

0c 4b 7c 3c

0c 0c 1d 0d

0c 0c 0e 0d

6a 7a 10a 9ab

4b 6a 9a 10a

1c 7a 11a 10ab

0d 3b 10a 8b

0d 0b 1b 0c

P26

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

9a 14a 15a 16a

4b 11b 12b 11b

1c 8c 7c 7c

1c 2d 1d 1d

0c 0e 1d 0e

10a 14a 15a 16a

6b 11ab 12b 13ab

2c 14ab 17a 18a

0c 10b 15a 12b

0c 0c 0c 0c

T1

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

5a 9a 8a 8a

2b 7b 7a 5b

0c 5c 5b 1c

0c 2d 0c 0c

0c 0d 0c 0c

5a 8a 8a 8a

4b 7ab 6a 5b

4b 8a 8a 4b

0c 6b 7a 2bc

0c 0c 0b 0c

T3

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

6a 6a 11a 9a

3b 6a 10a 9a

1c 6a 8b 8a

0d 1b 2c 1b

0d 0b 0d 0b

6a 6a 11a 10a

4b 6a 9b 11a

3b 7a 10ab 11a

0c 7a 11a 10a

0c 0b 0c 0b

1134

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

4a 5a 8a 8a

1b 4a 5b 5b

1bc 2b 3c 3c

0c 0c 0d 1d

0c 0c 0d 0d

4a 5a 8a 8a

3ab 5a 7a 7a

2b 5a 8a 9a

0c 4a 7a 8a

0c 0b 0b 0b

P44M

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

3a 4a 4a 5a

1b 2b 3a 3b

0c 1b 2b 2c

0c 0c 0c 1cd

0c 0c 0c 0d

3a 4a 4a 5a

2a 3a 4a 6a

2a 4a 4a 5a

0b 2b 4a 3b

0b 0c 0b 0c

P53M

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

3a 6a 7a 7a

0b 4b 6a 6b

0b 2c 5b 4c

0b 0d 2c 0d

0b 0d 0d 0d

3a 6a 7a 7a

4a 6a 7a 7b

1b 6a 7a 9a

0c 2b 7a 9a

0c 0c 0b 0c

P57M

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C

3a 5a 8a 9a

0b 3b 3b 4b

0b 1c 3b 3bc

0b 0d 1c 1cd

0b 0d 0c 0d

4a 5a 7a 9a

2b 4b 7ab 7ab

2b 3b 5b 5b

0c 2c 5b 6b

0c 0d 1c 0c

z

Dose of B. carinata pellets employed (milligrams of pellet per 9-cm plate). Values are means (n = 4). In temperature rows for each time of exposure, means followed by a different letter are statistically different (rmANOVA (P < 0.05) followed by multiple comparison test with Bonferroni’s corrections (P < 0.05)). y

four-way rmANOVA indicated significant effect of the factor time: F = 2,919.62; P = 0.000, as well as significant interactions between time and the other factors. In the same way, the results indicated significant effects of factors dose: F(4) = 4,818.74, P < 0.001; temperature: F(3) = 2,137.54, P < 0.001 and isolate: F (6) = 503.46, P < 0.001, as well as significant interactions among all of them. Then, means comparasion of doses were performed for each isolate and temperature (Table 1). After 3 days of exposure, mycelial growth rates of the isolates were significantly inhibited with the doses 6, 12 and 24 mg of B. carinata pellets, when they were compared with the control (dose 0) and at the four temperatures assayed. However, the inhibitory effect of the lowest dose (3 mg) was significant for all isolates only at 15 ◦ C and 30 ◦ C (except the isolate T3); and the effect was significant for only 6 of the isolates at 20 ◦ C and 25 ◦ C. With increasing doses of pellets, growth rate inhibition increased until the highest dose (24 mg), that resulted in a complete inhibition of the growth at the four temperatures. The inhibitory effect of the doses 3, 6 and 12 mg decreased between the third and the sixth

day of exposure, but the decrease of the effect was more noticeable at 25 ◦ C and 30 ◦ C. The dose 12 mg had a fungistatic effect at 15 ◦ C, while the dose 24 mg had a fungitoxic effect at all the assayed temperatures, except for the isolates P11, P21 and P57 M at 25 ◦ C. In the test of germination of chlamydospores the patterns of germination with different doses were similar at 20 ◦ C, 25 ◦ C and 30 ◦ C. However, the number of chlamydospores recovered in control plates at 15 ◦ C was significantly lower than the number expected and the pattern of germination was different from that observed with the other incubation temperatures. The combined effect of dose (X1 ) and degree-days of incubation (X2 ) on the percentage of germination of chlamydospores (Y) was examined with a stepwise multiple regression analysis. The model was Y = 0.281 − 0.021X1 + 0.003X2 (P < 0.001, R2 = 0.458). When this first model was expanded to include the interaction of dose and degree-days as a third independent variable, the model Y = 0.277 − 0.023X1 + 0.004X2 was also significant (P < 0.001) and R2 increased to 0.507 (Fig. 1). The percentage of germination was pos-

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Fig. 1. Effect of volatiles released by different doses (3, 6, 12 and 24 mg) of Brassica carinata pellets on the germination of chlamydospores of Phytophthora nicotianae with increasing degree-days accumulated. Degree-days was determined by the formula: (incubation temperature −12) × number of days of incubation.

itively related to degree-days, but negatively to the dose of pellet. Semi-partial correlations of coefficients indicated that 45.6% of the total variance in the percentage of germination was explained by the dose, while the degree-days explained 19.71% of that variance.

3.2. Inhibition of germination of chlamydospores in soil The effects of B. carinata pellets on germination of the chlamydospores added to the soil are presented in Table 2. On the one hand, for the chlamydospores in soil from the bottom of the containers and exposed to volatile and soluble compounds released by the lowest dose (BF3), significant differences in germination were observed between constant temperatures (H(3) = 64.73; P < 0.001) and between fluctuating temperatures (H(2) = 39.51; P < 0.001) (Table 2). The highest values of germination were achieved at 25 and 30 ◦ C. However, with the highest dose (BF6) the inhibition of the germination was total with either constant or fluctuating temperature, except at 25 ◦ C, where low percentages of germination were observed (Table 2). On the other hand, for chlamydospores in soil from inoculum bags, which were only exposed to the volatile compounds released into the headspace of containers, significant differences in germination between temperatures for the same dose were also observed (Table 2). When the soil did not interfere with the release of volatiles in the treatments “pellets without soil”, the percentages of germination were lower than when the pellets were incorporated into the soil (pellets with soil) for all temperatures and doses assayed. The highest percentages of germination were found at 25, 30 ◦ C and with the regime of temperature T2. However, when

the temperatures were extreme (15 ◦ C or T3) the percentages of germination were reduced. 3.3. Bioassay with pepper plant after soil biofumigation The effect of the two doses of B. carinata pellet assayed on survival of P. nicotianae inoculum in this experiment was evident. At the end of the 4-week biofumigation process under controlled conditions, the propagules of P. nicotianae were not detected in soils treated with any of the doses of pellet (BF3 and BF6), while in control treatment the recovered inoculum was 26 ± 25 CFU g−1 of dry soil. This difference was statistically significant (H(2) = 10.455; P = 0.005). Furtheremore, in the bioassays with pepper plants, no diseased plants were recorded in the biofumigated soils (BF3 and BF6), but the percentage of diseased plants in control treatment (C) was 82.5 ± 20.6%. The Kruskal-Wallis test indicated significant differences between biofumigated and control treatments (H(2) = 10.507; P = 0.005). 4. Discusion In the present work, the effect of the volatile compounds released by different doses of B. carinata pellets on the mycelial growth and the germination of chlamydospores of P. nicotianae was studied under different temperatures. The inhibition effect on the mycelial growth was significant for all the doses assayed after 3 days of exposure. The highest dose of pellet tested inhibited mycelial growth of all the isolates regardless of temperature, but the inhibition effect decreased between the third and the sixth day of exposure with lower doses of pellets,

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Table 2 Inhibition of the chlamydospores germination of P. nicotianae exposed to all the compounds released into the soil solution (volatile + soluble) during the biofumigation process or only to the volatile compounds generated during the process at different constant or fluctuating temperatures. Per compounds generated, germination of propagules (%)x Pellets with soily z

Pellets without soil

Dose

Temperature

Volatile (Inoculum bag)

Volatile + soluble (Inoculum from the bottom of the container)

Volatile (Inoculum bag)

BF3

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C T1 [14–19 ◦ C] T2 [27–33 ◦ C] T3 [30–40 ◦ C]

74 ± 7 b 60 ± 10 c 66 ± 8 bc 100 ± 10 a 49 ± 10 b 82 ± 12 a 8 ± 18 c

0±1 c 5±4 b 30 ± 5 a 24 ± 9 a 1±2 b 6±3 a 1±3 b

3±4 c 28 ± 5 b 47 ± 7 a 56 ± 20 a 0±1 b 73 ± 15 a 2±7 b

BF6

15 ◦ C 20 ◦ C 25 ◦ C 30 ◦ C T1 [14–19 ◦ C] T2 [27–33 ◦ C] T3 [30–40 ◦ C]

1±2 d 6±4 c 39 ± 6 b 62 ± 19 a 0±0 b 46 ± 10 a 0±0 b

0±0 b 0±0 b 2±2 a 0±0 b 0±0 a 0±0 a 0±0 a

0±0 a 0±0 a 0±0 a 0±1 a 0±0 a 0±0 a 0±0 a

Rates of B. carinata pellets: BF3 = 64.5 mg per container = 3 t ha−1 ; BF6 = 129 mg per container = 6 t ha−1 . Pellets were added into inoculated soil with P. nicotianae (500 chlamydospores g−1 of dry soil) situated on the bottom of the container. x Values are means ratios ± SD (n = 4). In column, for each dose and regime of temperature (constant or fluctuating), means followed by a different letter are statistically different (Kruskal–Wallis test (P < 0.05), followed by Mann–Whitney comparison test with Bonferroni correction (P < 0.01)). z

y

and this decrease varied depending on the temperature and the isolate. Although the isolates from Murcia showed growth rates lower than isolates from Extremadura, the inhibitory effect of volatile compounds was similar in both groups of isolates. In general, the decrease of inhibition effect was more noticeable at 25 ◦ C, the optimal temperature for mycelial growth of P. nicotianae (Erwin and Ribeiro, 1996). In a previous assay with 21 isolates of P. nicotianae, the inhibition effect of B. carinata pellets at 25 ◦ C also decreased along the time in all the isolates, with very low inhibition or no inhibition in most of them after 6 days of exposure (Morales-Rodríguez et al., 2014). Although the concentration of volatiles released, depending on time and temperature, was not monitored in the in vitro trials, the effects observed on mycelial growth suggest that release of ITCs is quick after hydrating the pellet and their activity is short in duration, no longer than 3–6 days. Price et al. (2005) found that concentration of allyl isothiocyanate (AITC) decreased with increasing time regardless of temperature, detecting the hightest concentration 4–8 h after hydrolisis and significant decreases at 24 h. Recently, Neubauer et al. (2015) confirmed these results reporting notable decrease of AITC concentration in soil 48 h after application of B. juncea seed meal. Similar results were found by Borek et al. (1994), Brown et al. (1994), Morra and Kirkegaard (2002) and Petersen et al. (2001). In the plate tests, volatiles released by pellets delayed germination of chlamydospores compared to control for all doses assayed, but none of the doses was lethal at all the temperatures of incubation. Thereafter, the same dose was more effective in inhibition of mycelial growth than in inhibition of chlamydospores germination. Previously, some authores have reported that mycelium of Rhizoctonia solani, Fusarium oxysporum var radicis f.sp lycopersici, Sclerotinia sclerotiorum and S. cepivorum, was more sensitive to biofumigation than fungi spores (Kurt et al., 2011; Smolinska and Horbowicz, 1999; Yulianti et al., 2006). The model used to predict chlamydospore germination indicates that dose had the greatest impact on germination and then, with higher doses of pellets the onset of germination was delayed. Consequenty, less degree-days were required for germination with lower doses. The patterns of germination at 20 ◦ C, 25 ◦ C and 30 ◦ C clearly evidence the effect of dose and degree-days. However, the appreciation of the real effect

of the dose at 15 ◦ C was difficult because the degree-days accumulated at this temperature was insufficient for germination of all the viable chlamydospores in control plates. Lutz et al. (1991) demostrated that the germination of P. nicotianae chlamydospores was correlated directly with accumulation of heat units from 0 to 150◦ -days, selecting a threshold temperature of 12 ◦ C since below this temperature heat units did not accumulate. Moreover, they also found that heat treatments at 21–33 ◦ C for incubation periods 2–8 days were more effective in increasing propagule germination than longer heat treatments at lower temperatures. Chlamydospores in the inoculum bags were only exposed to the volatiles released into the headspace of the containers. The highest rate (6 t ha−1 ; BF6) in the control containers inactivated the inoculum regardless of the temperature of incubation. When pellets were mixed with the soil, the effectiveness of both rates decreased, but part of the chlamydospores was inactivated. Thereafter, the losses of ITCs from soil due to volatilization results in high concentration in the headspace, enough to inactivate part of the inoculum. Several factors influence the ratio of volatilization of ITCs. The volatility of ITCs decreases with increasing molecular weight (Sarwar et al., 1998), so that AITC, with an aliphatic chain, is more volatile than aromatics. Price et al. (2005) found that the concentration of AITC in the air above the soil was higher above a sandy soil low in organic carbon than above a clay soil with higher organic matter content. It has also been demostrated that increasing soil temperature will increase the volatilization of AITC (Price et al., 2005). On the other hand, covering the soil reduces diffusion of AITC from the soil, since in covered soil the concentration of AITC was over three times that was measured in uncovered soil (Price et al., 2005). In our trials of inhibition of chlamydospores germination in soil, the application of a double commercial rate (BF6) of pellets into soil was effective regardless of the temperature (constant or fluctuating) of incubation, inactivating most of the chlamydospores in the amended soil. However, the commercial rate (3 t ha−1 ; BF3) was less effective and the number of viable chlamydospores after treatment was significant, specially when incubation was at 25 ◦ C and 30 ◦ C. In chlamydospores exposed only to the volatiles (inoculum bags) the effect of temperature of incubation was similar. This suggests that the treatments were more effective with low tem-

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peratures (15 ◦ C and 20 ◦ C), in contrast with previous results. Price et al. (2005) measured AITC concentration in the soil air and found that the maximum concentration at 30 ◦ C was reached after 4 h, and it was twice the maximum concentration at 15 ◦ C, that was reached after 8 h. AITC concentration decreased with time, and there were not differences between 30 ◦ C and 15 ◦ C after 24 h. Thereafter, the low percentages of germination of chlamydospores with low temperatures observed in our trials are not probably related to the effectiveness of the pellets, but to the insufficient number of degree-days accumulated during the incubation at 15 ◦ C and 20 ◦ C. In fact, the degree-days accumulated during the biofumigation of treatments and the posterior analysis on plate for quantification were 47, 82 and 54 for 15 ◦ C, 20 ◦ C and T1, respectively, in contrast with 117 and 152◦ -days for 25 ◦ C and 30 ◦ C, respectively. In general, the same rate was more effective when chlamydospores and pellets were incorporated into the soil than when chlamydospores were in the inoculum bags. In the first case, chlamydospores were in direct contact with the pellets and exposed to the volatile and soluble compounds released by the biofumigant. ITCs are very reactive compounds and their toxicity is due to reaction with sulphur containing groups in proteins (Brown and Morra, 1997). Besides ITCs, numerous other sulfurcontaining products have been identified as secondary products of glucosinolates in Brassica tisues (Gamliel and Stapleton, 1993; Vig et al., 2009; Wang et al., 2009), which may have influenced in the effectiveness of the pellets. Synergistic effects have already been demostrated by combining 2-propenyl ITC (AITC) and dimethyl sulfide (non-glucosinolate derived) (Bending and Lincoln, 1999). Kurt et al. (2011) found that the effectiveness of different aliphatic and aromatic ITCs against mycelial growth or carpogenic germination of S. sclerotiorum depended on whether they were used as vapour or contact phase. Warmington and Clarkson (2016) demostrated that effectiveness of several biofumigant crops for inhibiting carpogenic germination of S. sclerotiorum varied depending on whether the volatiles released were in direct contact with the sclerotia or in the vapour phase. Germination values obtained with low fluctuating temperatures (T1) were similar to those with constant low temperature (15 ◦ C). The lowest germination values regardless of the rate were obtained with the highest fluctuating temperature (T3 = 30–40 ◦ C) that simulated conditions of solarization in soil (Lacasa et al., 2010). In these conditions, temperature is the main inactivation factor, as indicate the reduced number of chlamydospores recorded in the unamended controls. Decreases in densities of P. nicotianae after soil solarisation have been previously reported (Juarez-Palacios et al., 1991; Coelho et al., 1999; Lacasa et al., 2015; RodríguezMolina et al., 2016). Coelho et al. (2000) determined the effect of cycling temperatures and cabbage amendments on the thermal inactivation of chlamydospores of P. nicotianae. Both rates of B. carinata pellets, the recommended commercial rate (BF3) and the double of this (BF6), reduced populations of P. nicotianae below the limits of detection of our bioassay (<2 CFU g−1 of soil) and totally controlled the disease on pepper. In principle, the best assay for evaluating the efficacy of the disinfestation method is growing the crop itself or a bioassay with a sensitive or susceptible plant (Termorshuizen and Jeger, 2014). Bowers and Locke (2004) reported that low or undetectable (<0.04 CFU cm−3 of soil) surviving population of P. nicotianae was able to cause disease on periwinkle in the greenhouse trials. In the present work, the experiment was carried out with autoclaved soil, without any interference of soil microorganism in the effectiveness of biofumigation. In a recent study, Warmington and Clarkson (2016) found that incorporating Biofence into pasteurized compost, at the same rate than our BF3, significantly reduced the carpogenic germination of Sclerotinia sclerotiorum sclerotia compared with the untreated control.

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