extfieldsofstudy: Medicine Chemistry

text:
Supplementation of Non-Starch Polysaccharide Enzymes Cocktail in a Corn-Miscellaneous Meal Diet Improves Nutrient Digestibility and Reduces Carbon Dioxide Emissions in Finishing Pigs

Simple Summary The objective of the present study was to investigate the effect of the non-starch polysaccharide enzymes cocktail (NSPEC) on growth performance, nutrient digestion and gas emissions on finishing pigs. The addition of the NSPEC into a corn-miscellaneous meal diet improved feed conversion ratio and increased the apparent total tract digestibility of dry matter, neutral detergent fiber, acid detergent fiber, crude protein and gross energy of the finishing pigs. Furthermore, the digestible energy of the diet was also increased by the NSPEC supplementation in the diet. In addition, the inclusion of NSPEC in the corn-miscellaneous meal diet reduced carbon dioxide emissions of a finishing pig house. The accumulation of manure could increase the release of ammonia in a pig house. Abstract This study was carried out to evaluate the effect of the addition of the non-starch polysaccharide enzymes cocktail (NSPEC) on growth performance, nutrient digestibility and gas emissions in a corn-miscellaneous meal-based diet for finishing pigs. The NSPEC is a combination of cellulase, xylanase, β-glucanase, β-mannanase, α-galactosidase and pectinase optimized by assessing the in vitro dry matter digestibility (IVDMD) of corn-miscellaneous meal diet using an in vitro method of simulating digestion in the stomach and intestine of growing pigs. Growth performance and apparent total tract digestibility (ATTD) of nutrients and energy were measured. The gas concentration of ammonia, carbon dioxide, nitrous oxide and methane in the environmental assessment chambers were determined. The gas detecting period was divided into three frequencies of manure removal of every 1d, 2d and 3d. The addition of NSPEC into the corn-miscellaneous meal diet decreased feed conversation rate (FCR) and increased the ATTD of dry matter, crude protein, gross energy, neutral detergent fiber and acid detergent fiber of pigs (p < 0.05). The digestible energy was also improved (p < 0.05) significantly by NSPEC supplementation in the diet. Furthermore, the supplementation of the NSPEC reduced (p < 0.05) carbon dioxide concentration in the chambers. The ammonia emissions were significantly increased according to average 1d, 2d and 3d manure removal procedures (p < 0.01). These results indicated that the inclusion of optimal NSPEC in a corn-miscellaneous meal diet improved growth performance, nutrient digestibility and reduced carbon dioxide emissions on finishing pigs. The accumulated manure could increase the release of ammonia in a pig house.

test diets are summarized in Table 1. The feed was manufactured in dry mash form and formulated to meet or slightly exceed the nutritional requirements of finishing pig as recommended by the National Research Council [22]. The methodology for screening the NSPEC in a diet using an in vitro method of simulating digestion in the stomach and intestine of pigs was developed in our lab [5]. The optimal NSP enzyme cocktail in the corn-miscellaneous meal diet was 1002 U/kg cellulase, 18,076 U/kg xylanase, 1376 U/kg β-glucanase, 14,765 U/kg β-mannanase, 337 U/kg α-galactosidase and 138 U/kg pectinase in the corn-miscellaneous meal diet. The NSPEC was screened by assessing the IVDMD of the corn-miscellaneous meal diet using an in vitro method of simulating digestion in the stomach and intestine of growing pigs.
Sixteen crossbred barrows (Duroc × (Landrace × Large White)); initial body weight of 117.89 ± 0.85 kg; Beijing Breeding Swine Center, Beijing, China) were randomly allotted into 2 dietary treatments and each treatment had 8 replicates which were randomly divided into 2 environmental control chambers and each chamber had 4 pigs. Each environmental control chamber fed 4 pigs which were housed in stainless steel metabolic cages (1.2 m × 1.5 m). The daily feed allowance was calculated at 3.5% of the initial weight of pigs. Pigs were fed one-half of the daily feed allowance each at 8:00 and 15:00 per day and provided ad libitum access to water during the entire experimental period. After a 5d adaption period and 3 d feces collection period, feces were collected via grab sampling and stored at −20 • C immediately after collection. To avoid differences between the environmental control chambers, the pigs of the CT group and NSPEC group were exchanged between the two experimental periods, and each period had a 3d gas detecting period. The 3d gas detecting period was divided into 3 frequencies of manure removal of every 1d, 2d and 3d.

Chemical Analysis
At the completion of the experiment, feces samples were thawed, mixed and oven-dried at 65 • C for 96 h. The feed and feces samples were ground through a 0.5 mm sieve in a centrifugal grinder before analysis. All samples of diets and feces were analyzed for dry matter (DM, method 930.15) and crude protein (CP, method 990.03) following the procedures outlined by the Association of Official Analytical Chemists [23]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using filter bags and fiber analyzer equipment following a modification of the procedures [24]. Samples of the feed were analyzed for extract ether (EE; method 954.02) and ash (method 942.05) [23]. The gross energy (GE) in the diets and feces were determined using an adiabatic bomb calorimeter (Model 6400; Parr Instrument, Moline, IL). All the analyses were performed in duplicate.

Calculations
The ATTD of DM, CP, NDF, ADF and GE were calculated in the diets according to former reported equations [25]: where ATTD is the apparent total tract digestibility of gross energy (%), N i is the total intake of DM, CP, NDF, ADF and GE in the feed, and N o is the total fecal output of DM, CP, NDF, ADF and GE.

Measurements of Gas Concentrations
The concentration of gas in all the chambers was measured with a Photoacoustic Field Gas-Monitor INNOVA 1412 (LumaSense Technology, Santa Clara, CA, USA). Simultaneous measurement of NH 3 , N 2 O, CH 4 and CO 2 was carried out throughout the entire experiment. The gas emissions expressed per day and per livestock unit were corrected to 500 kg body weight. This system was designed to continuously monitor incoming and exhaust concentrations of gases, including control chamber ventilation volume, temperature and humidity to ensure the stability of the cabin environment [26].
A TH100 thermal gas mass flow meter was placed at the ventilator vent to monitor the ventilation environmental controlled chamber in real time. All gas samples were collected in the middle of the intake and exhaust ducts. At the time of gas collection, the gas sampling system continuously delivered the collected gas to the gas detector. Each cabin gas sample was measured 5 times in a sequential manner (every time for 1 min) and it was continuously measured for 24 h.
Egas: daily gas emissions of per pig (g/pig/d); D: ventilation rate at house temperature and pressure, (L/min); Co: concentration of exhaust house ventilation air (ppm); Ci: gas concentration of incoming house ventilation air (ppm); N: the number of pigs in the house; T: ventilation time (24 h); M: molecular weight of the gas; t: the temperature in the house; Thereafter, the daily emissions were calculated for each series of measurements and expressed per pig and per livestock unit (LU) that equals 500 kg body weight.

Statistical Analysis
The UNIVARIATE procedure (SAS Version 9.2, SAS institute Inc., Cary, NC, USA) was used to confirm the homogeneity of variance and also analyze for outliers, but no outliers were identified. The normality of the data distribution was also tested prior to the final comparison by SAS. Growth performance and nutrient digestibility data were analyzed by Student's t-test. Diet was a categorical variable. Frequency of manure removal was treated as an ordinal variable. According to a completely randomized design for gas emissions, the diet, frequency of manure removal and diet × frequency of manure removal interaction were treated as fixed effects, whereas animals, chambers and periods were treated as random effects by using the MIXED procedure of SAS. The differences were considered significant if p < 0.05 and were considered a trend if the p-value was between 0.05 and 0.10.

Growth Performance and Nutrient Digestibility
The supplementation of the NSPEC in corn-miscellaneous meal diet had no effect on the average daily gain (ADG) and feed intake (FI) in finishing pigs (Table 2). However, the pigs fed the NSPEC diet had lower feed conversion rate (FCR) than the CT diet (p < 0.05). Compared with the CT group, the inclusion of the NSPEC improved ATTD of DM by 2.4%, CP by 2.84%, NDF by 4.9% and ADF by 5.93% during the overall period (p < 0.05). In addition, the ATTD of the GE and DE were also improved by the NSPEC supplementation (p < 0.05).  according to different frequency of manure removal, respectively. The NH 3 emissions were significantly increased according to average 1d, 2d and 3d manure removal procedures (p < 0.01). The evolution of N 2 O and CH 4 emissions showed no particular trends throughout the experimental period. No differences were observed for the N 2 O and CH 4 emissions compared the NSPEC group with the CT group. However, the ADC (average daily gas concentration) of CO 2 was significantly lower (p < 0.05) in the NSPEC group than in the CT group. Furthermore, the ADEU of CO 2 was remarkably decreased or showed a downward trend (p = 0.06) in pigs fed a diet supplement with NSPEC.

Discussion
In the present study, corn-miscellaneous meal diet with high fiber content was mainly composed of corn, soybean meal, wheat bran, cottonseed meal and sugar beet pulp. The xylan content accounts for the main part of the NSP composition of these feed ingredients. In addition, the mannan content also occupies a large proportion. Therefore, the enzyme cocktail mainly included xylanase and β-mannanase in our study. The pigs fed the NSPEC diet had a relatively lower FCR than the pigs fed the CT diet. The reduced FCR might result from the supplementation of enzyme cocktail in the diet improving the relative NSP digestibility. Our results are also in agreement with a previous study which reported that non-starch polysaccharide-degrading enzymes supplementation improved the FCR of growing pigs fed diets with multi-enzyme [27]. In addition, some studies observed that a complex of non-starch polysaccharide-degrading enzymes could improve the growth performance of the weaned piglets and growing-finishing pigs [28][29][30][31]. However, some studies failed to observe a positive effect of enzyme cocktail supplementation on growth performance of pigs [32]. The apparent contradictions in the effectiveness of multi-enzyme supplementation on growth performance among studies may be mainly attributed to the differences in age of the pigs and the composition of diets used. In addition, the enzyme source and the combination of various NSP enzymes may also exert a different effect on growth performance.
The NSPEC supplementation increased the ATTD digestibility of DM, NDF and ADF by 2.4%, 4.9% and 5.93%, respectively in growing pigs. The ATTD of CP, GE and DE were also improved when the NSPEC was added in the diet. These results were consistent with a previous study which reported that multi-enzyme supplementation increased nutrient digestibility in pigs [33,34]. For example, Li et al. reported an increase in DM, GE and CP in growing pigs fed a diet supplemented with an amylase, protease and xylanase blend compared to a corn-soybean meal-based diet [35]. The improvement in nutrient digestibility in our study indicated that the NSPEC exerted its beneficial effects on nutrient digestibility of the finishing pigs, probably through first breaking down the plant cell wall structure and then releasing the nutrients for use by the pig [36].
The level of gas concentrations is the balance between the production by the animals' respiration and/or the manure and the evacuation by the exhaust fans. All the NH 3 , N 2 O, CH 4 and CO 2 emission patterns reflect the increase of feed intakes, the higher metabolism of animals and the accumulation of manure during the entire experiment. We found that the NH 3 emissions were significantly increased according to average 1d, 2d and 3d manure removal procedures. The N 2 O, CH 4 and CO 2 emissions showed an upward trend with the accumulation of manure. A previous study has reported that accumulated manure could increase the release of gases by a pig house [37]. Furthermore, manure removal frequency has been proposed to serve as an efficient means to reduce the emissions of harmful gases from pig buildings. Some researchers found that cumulative CH 4 emissions were shown to be lower by 16% and N 2 O emissions remained the same when manure was removed three times a week instead of only one time in growing pigs [38]. The NH 3 emissions data demonstrated that there were no significant differences observed between the NSPEC and CT group. The results of this experiment are consistent with a previous study in which exogenous enzyme supplementation in cereals increased ammonia emissions on finisher pigs [39]. However, it was observed that enzyme supplementation decreased ammonia emissions in wheat based diets, while in barley-based diets enzyme supplementation increased ammonia emissions in pigs [40,41]. These opposing results could be elucidated by the difference of the NSP composition of these cereals. For instance, the NSP fraction of barley mainly contains a mixture of β-glucans and arabinoxylans, while arabinoxylans are the main NSP component of the wheat [42]. In pig houses, the formation of nitrous oxide originates only from manure. N 2 O is an intermediate product and its formation mainly takes place during incomplete nitrification and denitrification processes [43]. The supplemental NSPEC in the diets had no significant effect on N 2 O emissions for fattening pigs. There were a few data on N 2 O emissions in the literature, which accounts for approximately 10% of the NH 3 emission mass [44]. Some authors suggested that reduced NH 3 emission strategies could also limit N 2 O emissions since NH 3 is the precursor of the formation of N 2 O [45]. Another greenhouse gas in pig houses is methane. CH 4 emissions mainly result from enteric fermentation in the gut [46]. In our study, the levels of CH 4 emissions were not altered after the NSPEC treatment. However, some authors observed a tendency for higher CH 4 emissions with xylanase supplementation [47]. Although the amount of CH 4 emissions from pig houses is thought to be very little, its emissions are closely related to the fiber content in the diet [48,49].
The carbon dioxide emissions from pig houses mainly originate from two sources including exhalation by pigs and release from manure. Several authors measured a 25% reduction in CO 2 emissions from pig breathing, as a consequence of reduced pig activity [50]. CO 2 release from manure was ignored for many decades [51,52]. However, some researches indicated that the levels of CO 2 emissions from manure have been evaluated to be 4-5% of the entire amount of CO 2 exhaled by livestock [53]. The CO 2 emissions of manure principally comes from three sources, namely: (1) the rapid hydrolysis of urea into NH 3 and CO 2 catalyzed by the urease; (2) the aerobic degradation of organic matter; (3) the anaerobic fermentation of organic matter into intermediate product such as volatile fatty acids (VFAs), CH 4 and CO 2 [54]. The third process is usually regarded as the principal source of CO 2 [55]. The present study indicated that the ADC of CO 2 in the supplemental NSPEC group was significantly lower than the CT group. These results may correspond to the increase of the ATTD of NDF and ADF in the NSPEC supplementation group. We have demonstrated for the first time that supplementation with NSPEC could reduce CO 2 emission from swine houses according different frequencies of manure removal. Nevertheless, further investigations have to be carried out to clarify detailed gas emission mechanisms under supplemental NSP enzyme cocktail. We only showed that the optimal NSPEC supplementation in corn-miscellaneous meal-based diet could reduce CO 2 emissions and increase NH 3 emissions with the manure accumulation for finishing pigs. Therefore, further studies need to be carried out in order to evaluate the effect of the optimal NSPEC on NH 3 and GHG emissions in fattening pigs fed other diets.

Conclusions
In conclusion, the supplementation of NSPEC in a corn-miscellaneous meal-based diet could improve pigs' growth performance. Furthermore, the beneficial effects of the NSPEC supplementation in pigs' diet on nutrient digestibility improvement and CO 2 emission reduction are more obvious compared with the CT diet. Therefore, supplemental NSPEC could promote swine production efficiency and improve the feeding environment of fattening pigs. In the meantime, the accumulated manure could increase the release of ammonia in pig houses. Funding: This work was financially supported by the National Natural Science Foundation (31702119) and the Agricultural Science and Technology Innovation Program (ASTIP-IAS07) in China.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Photodynamic inactivation of Botrytis cinerea for a safe pest management of grapevine

Botrytis cinerea is a necrotic fungal plant pathogen responsible for the gray mold disease on more than 200 crops, including grapevine. Due to its genetic plasticity, this fungus presents a strong resistance to a large spectrum of fungicides. Thus, new fighting strategies against B. cinerea are urgently needed. In this context, antimicrobial photodynamic treatment (APDT) was considered. APDT involves the use of a photosensitizer that generates reactive oxygen species upon illumination. Tetra-4-sulfonatophenyl porphyrin tetra-ammonium (TPPS) was tested on B. cinerea ; upon light exposure. 1.5 µM TPPS was shown to completely inhibit mycelial growth. A concentration of 12.5 µM TPPS was tested on three genetic background clones from Chardonnay, Merlot and Sauvignon, grown in vitro for 2 months. Treated root apparatus of the three backgrounds was able to increase thiol production as a molecular protection against photoactivated TPPS, leading to a normal phenotype as compared with control plantlets. Finally, two-month-old grapevine leaves were infected with four-day-old mycelium of B. cinerea pre-incubated or not with TPPS. The pre-treated mycelium was unable to infect the isolated leaves of any of the three grapevine varieties after 72 h growth when subjected to a 16 h photoperiod, contrary to untreated mycelium. These results on fungus, plantlets and Botrytis-grapevine leaves, in contact with a very low concentration of TPPS, suggest a strong potential of photo-treatment against Botrytis mycelium for future agricultural practices in vineyard or other cultures.

Introduction
The great challenge of agriculture is to produce sufficient food for the ever-growing world population.
Since the 60 s, to access this performance, agri-business practices that include excessive uses of pesticides and fertilizers are becoming the main cause of soil, water and air pollution, as well as loss of fauna and flora biodiversity (Carvalho 2017; Meftaul et al. 2019). Moreover, this industrial agriculture triggers major public health problems such as infertility, cancers and child malformations.
Aware of the need to reduce all these dramatic environmental damages, the European Union introduced the directive 2009/128/EC to reduce the use of pesticides.
To fight off plant competitors and pathogens, new approaches are necessary for safe practices in agriculture such as genetically modified plants, genetic improvements, as well as organic and integrated agriculture (Kiley-Worthington 1981;Key et al. 2008). The photodynamic treatment is a general and new concept with a large spectrum of applications for animal and plant cells, plant and animal pathogens, as well as microorganisms (Ben Amor and Jori 2000;Jori and Brown 2004;Donnely et al. 2008;Maisch 2009;Almeida et al. 2011;Jori 2011;Alves et al. 2015;Liu et al. 2015;Hamblin 2016;Kashef et al. 2017;Issawi et al. 2018a;Ambrosini et al. 2019). Thus, photodynamic treatment could represent an innovative and powerful strategy to fight off plant competitors and pathogens in future agricultural practices (Issawi et al. 2018a). One of the key actors of APDT is a molecule called a photosensitizer (PS). When irradiated with light, this molecule produces reactive oxygen species that are toxic for cells (Dai et al. 2009). By contrast, most PSs present low levels of cytotoxicity or genotoxicity in the dark (Luksiene et al. 2004;Donnelly et al. 2008). Furthermore, biological applications are best conducted with water-soluble PSs which are ideally prone to quick photodegradation, thus avoiding a buildup of toxicity. PSs are classified in many groups such as porphyrins, chlorins, coumarins, furocoumarins, phthalocyanines and phenothiaziniums. Porphyrins and chlorins such as chlorophyllin, have been shown to be very effective against bacteria on kiwi leaves tested in vitro (Jesus et al. 2018;Glueck et al. 2019). Coumarins, furocoumarin and phenothiazinium were shown to be active against the plant-infecting fungi Colletrichum acuratum and Aspergillus nidulans (De Menezes et al. a, b;2014;Fracarolli et al. 2016). Finally, when tested on Citrus sinensis petals and leaves, methylene blue was able to kill the Colletotrichum abscissum fungus and proved to be harmless to plant organs; in addition, this treatment did not induce any secondary resistance (Gonzales et al. 2017) Previous research works explored the in vitro phenotypical and molecular responses of Arabidopsis and tomato plantlets to the photodynamic stress induced by an exogenous supply of PS (Guillaumot et al. 2016;Issawi et al. 2018b). The cationic tetra (N-methylpyridyl) porphyrin, either free base or zinc-complexed, tested at 3.5 µM, inflicted harmful effects on both 14-day-old Arabidopsis and tomato plantlets. Nevertheless, while Arabidopsis plantlets were killed, tomato plantlets could be rescued after a 14 day-treatment (Guillaumot et al. 2016). Surprisingly, the anionic porphyrin tetra-4-sulfonatophenylporphyrin tetra-ammonium (TPPS) did not provoke any harmful effect on both plantlets even at concentrations as high as 50 µM (Guillaumot et al. 2016;Issawi et al. 2018b). With the aim to develop APDT for agriculture applications, TPPS could be a good candidate because of its low toxicity for plants. Moreover, TPPS remains negatively charged in many chemical environments even under acidic pH and does not aggregate in solution, allowing it to easily permeate cells through cell walls and membranes Issawi et al. 2019). Therefore, we hypothesized that TPPS could be a good PS candidate to kill the plant pathogen B. cinerea and be a safe option for grapevine (Vitis vinifera L.) explants.
B. cinerea is frequently responsible for drastic reductions in crop yields at harvest and for reducing wine quality (Rosslenbroich and Stuebler 2000;Williamson et al. 2007). This fungus displays very strong resistance to many fungicides, due to its genetic plasticity which confers its diversity in morphology, mycelial growth, sporulation and virulence (Movahedi and Heale 1990;Yourman et al. 2001;Valiuskaite et al. 2010;Nakajima and Akutsu 2014;Nakamura and Iwai 2019). For the Integrated Pest management, the grapevine's susceptibility to B. cinerea can be considered an essential management indicator (Galet 1988;Dry and Gregory 1988;Marois et al. 1992;Kogan 1998;Dubos 2002;Fermaud et al. 2011). According to the classification proposed by Dubos 2002 andFermaud et al. 2011, Chardonnay and Sauvignon are highly susceptible to B. cinerea infection whereas the Merlot variety is more resistant. Furthermore, these three varieties are listed in the top 10 most cultivated and famous grapevines for wine production in the world (Anderson 2013).
Therefore, as a first and necessary step, TPPS was tested separately on the three grapevine backgrounds and on Botrytis mycelium with the aim of killing the plant pathogen without affecting plantlet growth and development. As a second and final step, grapevine leaves infected with Botrytis mycelium were tested with and without TPPS pre-treatment. Photosensitizer 5,10,15, porphyrin tetra-ammonium (TPPS) was purchased from PorphyChem (Dijon, France). The stock solution (1 mM) was prepared in distilled water and kept in the dark at room temperature for 2 weeks.

Botrytis cinerea culture
The B. cinerea strain (UBOCC-A-117017) used in this study was isolated from infected tomatoes and provided by Dr Amélie Weill (Université de Bretagne Occidentale, Brest, France). The culture was maintained on potato dextrose agar (PDA). The growth curve of B. cinerea was performed as follows: a plug (0.6 cm diameter) of two-week old B. cinerea mycelium was placed in the middle of plates containing PDA medium supplemented with or without TPPS in the concentration range 0.5-3.5 µM.
TPPS was added to the PDA medium just after autoclaving (120 °C, 20 min). Plates were incubated at 22 °C, either in the dark or subjected to a photoperiod of 16 hours. Daily measurements of the diameter of the fungus were performed and reported to draw the growth curves.
Grapevine clone culture Chardonnay (clone 7535) was provided by Pr. Clément (Université de Reims, Champagne-Ardenne, France). Sauvignon (clone 379) and Merlot (clone 373) were provided by the Institut Français de la Vigne et du Vin (Bordeaux, France). Intermodal explants of grapevine were dissected and placed in glass tubes or jars containing half Chée and Pool medium, and 2% (w/v) sucrose-solidified medium (pH 5.9) for 2 months. TPPS was added to the medium after autoclaving. Chée and Pool medium was purchased from Duchefa Biochemistry (Haarlem, Holland). The cultures were then exposed to white light (photon flux density of 120 µmol.m − 2 .s − 1 ) for 16 hours and the temperature was maintained at 24 °C.

Infection of young leaves with B. cinerea
Two-month-old grapevine leaves and four-day-old mycelium plug (0.6 cm diameter) were used to perform the experiment. Mycelium discs were firstly incubated in 12.5 or 50 µM TPPS in the dark and gently stirred for 8 hours at 22 °C. Fungus discs were then placed on the upper leaf epidermis of the grapevine (Chardonnay, Merlot and Sauvignon). The co-cultures were monitored daily and photographed using a Leica stereomicroscope.

6
The MTT assay was performed to quantify the mitochondrial activity of mycelial cells. The fungal samples were frozen in liquid nitrogen and were ground to a powder. 1 mL of 0.1% (m/v) MTT solution was added to 150 mg of powder. Samples were left in the dark and stirred for 3 hours at room temperature. The suspension was centrifuged at 4,000 × g for 10 min. Supernatant was discarded and 1 mL of isopropanol was added to the pellet. Samples were vortexed and centrifuged at 4,000 × g for 10 min. Absorbance was read at 590 nm.

Determination of malondialdehyde content
Approximately 150 mg of fresh or frozen fungal material were ground in liquid nitrogen. 1.5 mL of 20% (w/v) TCA was added onto the powder. The mixture was centrifuged at 13,000 × g at 4 °C for 20 min. The supernatants were analyzed for their malondialdehyde (MDA) content as described by Hodges (1999) with minor modifications described in Issawi et al. (2018b).

Hydrogen peroxide quantification
The measurement of Hydrogen peroxide (H 2 O 2 ) was performed according to a modified procedure (Pandey 2013). 1 mL of extraction buffer (50 mM, pH 7.8) was added to fresh samples such as mycelium, plantlets or in vitro isolated leaves. The composition of the extraction buffer added to the mycelium samples was 1 mM EDTA, 1% (w/v) PVP, 10% (v/v) glycerol and 1 mM DTT. For the plantlet and the leaf samples, the extraction buffer was almost identical, however PVPP was used in the buffer, instead of PVP. All samples had been frozen in liquid nitrogen and prior to being grounded into a powder. Homogenates were centrifuged at 13,000 × g at 4 °C for 20 min. 335 µL of 0.1% titanium III sulfate (v/v) dissolved in a solution of 20% (w/v) H 2 SO 4 , and this solution was added to supernatants.
Absorbances were read at 415 nm and H 2 O 2 levels were expressed as nM.g -1 protein. Protein concentration was determined by Bradford assay using BSA as standard (Bradford, 1976).

Total Thiol Assay
Approximately 100 mg of fine powder has been obtained from the samples (fungus, plantlets or in vitro isolated leaves). After grinding in liquid nitrogen, 1 mL of 0.2 N HCl was added to the powder. A centrifugation at 13,000 × g for 20 min was performed. Afterward, 500 µL of the supernatant were neutralized with 400 µL NaOH (0.2 M) and 50 µL NaH 2 PO 4 (0.2 M). 700 µL of 0.12 M NaH 2 PO 4 , 6 mM EDTA and 0.1 mL of 6 mM dithiobis-2-nitro-benzoic acid (DTNB) were added to 200 µL of extract. A standard calibration curve was prepared by replacing the extract with 0, 5, 10, 25 and 50 µg/mL glutathione solutions (total volume 1 mL). Absorbance at 412 nm was read 5 min after the addition of glutathione or extract.

Statistical Analysis
All biological experiments were performed at least three times independently. Results were expressed as a mean ± SD (Standard Deviation). The data were analyzed by t-student test and one-way ANOVA using the PAST free software.

Mycelium growth
The effect of three increasing concentrations of TPPS was monitored on B. cinerea mycelium growth under dark and light conditions as described in the material and methods section. As the four curves obtained with or without TPPS in the dark did coincide within the standard deviation, we confirmed that, by itself, TPPS did not exhibit any cyto-and/or genotoxic activity against the fungus (Fig. 1a).
Under light and at a low TPPS concentration (1.5 µM), mycelium growth was completely inhibited (Fig.  1b). Furthermore, 1 µM TPPS significantly slowed down mycelium growth under light; however, after a 7 day-culture, the colony reached a size similar to that of the control (Fig. 1b). Therefore, the concentration of 1.5 µM of photoactivated TPPS has been chosen as the minimum fungicidal concentration (MFC). As shown by the growth curves in the dark and under light conditions, the fourth day of culture corresponded to the end of the exponential growth phase (Fig. 1). Thus, all further experiments were conducted with 4-day old mycelium Effect of photoactivated TPPS on hyphae morphology As mycelium growth was affected by photoactivated TPPS, it was decided to carefully look at the hyphae structure, using ESEM. It has been shown that stressed mycelium often produces spores and could show altered cell elongation (Schumacher 2017). As expected, photoactivated TPPS induced important phenotypic changes of the hyphae, compared with the control that showed very regular hyphae with a well-organized structure (Fig. 2a). In presence of 1.5 µM TPPS, hyphae were notably less organized, exhibited irregular shapes and produced some spores (Fig. 2a). Moreover, the TPPStreated hyphae presented a reduced width (1.36 µm) compared with the control (4.7 µm) (data not shown).

TPPS localization inside mycelial cells
According to the phenotypical effects observed in TPPS-treated mycelium under light, it was of interest to localize TPPS inside the cells. In tobacco plant cells, TPPS was also tested at 3.5 µM and it was the most effective porphyrin PS to induce cell death under a short light period of 5 hours and it was proven to mainly localize in the cell wall (Riou et al. 2014;Issawi et al. 2019). Thus, a similar localization of TPPS was expected in the fungal cells. Surprisingly, TPPS was found inside several cells of four-day-old mycelium (Fig. 2b). This intracellular localization could explain the very strong inhibitory effect of photoactivated TPPS on mycelial growth. As TPPS was located inside cells, this showed that it could cross the cell wall and accumulate in the cell cytoplasm. The multi-layer fungal cell wall is enriched in neutral sugars and proteins and poor in chitin and uronic acids, suggesting a neutral global charge of the cell wall (Cantu et al. 2009). Nevertheless, the Botrytis cell wall composition and its global charge remain controversial. We hypothesized that this neutrality could allow TPPS to cross the fungal cell wall which is completely different from the tobacco cell wall (Cantu et al. 2009;Issawi et al. 2017Issawi et al. , 2019.

Biochemical activities of TPPS-treated B. cinerea mycelium
To gain insight into the effect of photoactivated TPPS on the mycelium, biochemical assays linked to oxidative stress and cell metabolism activity, were conducted. As expected, increases in both H 2 O 2 and MDA contents were observed in treated mycelium compared to the control, demonstrating at the molecular level that the fungus was stressed (Fig. 3). Metabolic activity, linked to mitochondrial respiration, was monitored with the MTT assay. Indeed, under light, formazan production decreased in the treated hyphae compared with the control, thus explaining the fungal growth inhibition (Fig. 3).
While MDA indicated an increase in lipid peroxidation in the presence of photoactivated TPPS, the thiol content provided additional evidence that the fungus was not dead but only under a large amount of stress (Fig. 3).

TPPS and grapevine plantlets grown in vitro
Internodal explants from each variety: Merlot, Sauvignon and Chardonnay, were placed on 12.5 µM of TPPS for two months in growth chambers as described in the material and methods section. Each culture was examined during a period of 3-months and the explant sub-culture was performed from two-month-old plantlets. There was no phenotypical difference between the control and the treated plantlets after a one-month culture (Fig. 4). To confirm the absence of the phenotypical effect of photoactivated TPPS, we measured the thiol content in aerial and root organs of treated and control plantlets from the three varieties (Table 1). Thiols are powerful antioxidants and thus, their cellular content is a good indicator of an efficient response to oxidative stress. Roots from the three varieties, that were in contact with photoactivated TPPS, showed a significant increase in the total thiol content compared to the control (Table 1). At the aerial level, while no difference in thiol content was observed in Sauvignon and Merlot, there was a significant difference in the total thiol content measured in Chardonnay (Table 1). This could suggest that the Chardonnay variety is more sensitive to photoactivated TPPS than the two other backgrounds although no visible outcome could be seen at the phenotypic level (Fig. 4).

TPPS effect on Botrytis-infected leaves
The final interest of this work was to put together grapevine, Botrytis and TPPS with the expectations to kill the pathogen without disturbing plants. As a preliminary assay, before the development of a complete plant pathosystem, our antifungal photodynamic treatment was tested on isolated grapevine leaves from two-month-old plantlets. Experiments were conducted as described in the material and methods section, in the growth chamber. The infection was monitored for 72 h. Before 48 h, no change was observed in leaves from the three clones (data not shown). After 48 hours, the untreated B. cinerea started to invade the leaf surface of the three varieties. The growth of the mycelium, pre-treated with 12.5 µM of TPPS for 8 h, was not completely inhibited (Fig. 5). However, after a 50 µM TPPS pre-treatment, the growth of mycelium was totally inhibited on the leaf surface suggesting that the strategy described in this study worked efficiently.
ESEM analysis was conducted on infected leaves. In uninfected leaves, the structure was well-defined and organized (Fig. 6). However, in the infected leaves when Botrytis was not pre-treated with TPPS, the fungus completely invaded the leaves, making the structure unrecognizable (Fig. 6). By contrast, when B. cinerea was pre-treated with 50 µM of TPPS, ESEM pictures showed a leaf structure almost identical to that of the control confirming the macroscopic phenotype (Fig. 6). It was noticed for the Sauvignon variety, that spores were present in the pre-treated Botrytis suggesting that Sauvignon was more susceptible than the other two varieties.
For further investigation, biochemical assays were performed. H 2 O 2 quantification, linked to oxidative stress, was conducted for the three leaf conditions and the three varieties after infection or not with TPPS. After a 72 h treatment, a basal content of H 2 O 2 was detected in the three leaf varieties, very similar for Chardonnay and Sauvignon leaves and slightly lower for Merlot (Table 2). When the leaves were infected by a 4-day-old B. cinerea mycelium, the H 2 O 2 content significantly increased for the three varieties especially for Sauvignon (more than 3.5 fold higher than the control leaf) explaining its strong susceptibility to Botrytis (Table 2). B. cinerea is a necrophytic fungus that induces ROS production that contributes to plant cell destruction during its infection (Choquer et al. 2007).
Therefore, an increase in H 2 O 2 production in all leaf types in contact with Botrytis was expected.
Concerning the leaves infected by the fungus pre-treated with 50 µM TPPS for 8 h, no significant difference was observed between Chardonnay and Merlot leaves compared to leaves infected by the fungus. No signs of infection were observed after 72 h culture (Fig. 5 and Table 2). Botrytis cinerea, pre-treated with TPPS, was no longer able to induce a high production of H 2 O 2 in Sauvignon leaves ( Table 2). The H 2 O 2 content nearly reached the basal level detected in the control Sauvignon leaves ( Table 2). As a response to this H 2 O 2 production detected in the leaves induced by Botrytis, and to gain insight into the leaf's proper response, we measured the total thiol content in leaves that were subjected to the three different treatments. Thiols are well known antioxidants and are considered as a primary and strong defense to infection (Ulrich and Jakob 2019). For Chardonnay and Merlot isolated leaves, no significant difference was observed between the control, infected leaves and infected leaves pre-treated with TPPS (Table 2). A significant increase in thiol content was only observed for Sauvignon leaves between the control and the infected leaves. This result suggested that Sauvignon leaves were able to fight against the fungus infection with a thiol induced response (Table 2).
According to our H 2 O 2 and thiol content results and what has been previously published in literature, we confirm that Sauvignon was more susceptible to B. cinerea infection, compared to Merlot and Chardonnay (Dubos 2002;Fermaud et al. 2011). Nevertheless, we also confirm that our pretreatment with TPPS inhibited Botrytis growth on leaves for each variety. These results are promising for the development of APDT treatments in agriculture ( Fig. 5 and 6).

Discussion
B. cinerea is a very serious problem in a large variety of plants. This fungus is able to infect leaves, stems, flowers and fruit, causing severe damages and commercial losses in agriculture. In vineyards, the fungus induces several deleterious effects on both quality and quantity of vine production.
Despite all the damage it can cause, under specific weather conditions, its growth on grapes induces noble rot that gives rise to sweet wine.
However, the fight against this pathogen remains a daily struggle especially for fruit production in summer or fall. For more than 50 years, the use of specific fungicides has largely been envisaged and in that time, Botrytis had to find coping strategies. As a consequence, fungicide treatments gradually became inefficient, even the famous CuSO 4 solution also known as 'Bordeaux mixture' lost its effectiveness against fungal pathogens (Leroux et al 2002;Hahn 2014). Moreover, the copper divalent ion, also toxic for plants, contributed to soil pollution (Fernandes and Henriques 1991; Borkow and Gabbay 2005).
Therefore, new strategies against fungi are urgently needed. Wang et al. (2010) used naturally occurring eugenol (EC50 value of 235 µM for B. cinerea). This was found to mainly affect fungal mycelium growth rather than the germination of spores as shown in previous reports on fungicides, such as carbendazim and N-phenyl carbamates (Sherald et al. 1973;Suzuki et al. 1984).
In a similar way, Fleurat-Lessard et al. (2011) discovered a strategy whereby they investigated the potential of FeSO 4 and found that the sulfate anion determines the inhibition of mycelium growth in pathogenic fungi that is similar to B. cinerea at high concentrations (range of 0.5-20 mM). They also tested different iron salts and found that those with bromide, chloride and sulfate anions showed the best antifungal activity. In particular, the addition of an ammonium counterion to the sulfate moiety contributed to the inhibition of mycelium growth in the pathogenic fungus Eutypa lata, an ascomycete like B.cinerea. Therefore, taking these results from previous works into account (Fleurat-Lessard et al. 2011;Guillaumot et al. 2016;Issawi et al. 2018b;, we decided to test TPPS, a molecule that presents four external sulfonate groups linked with a tetrapyrrole ring. Indeed, TPPS with an ammonium counterion could be as an excellent antifungal candidate. Moreover, it was demonstrated that TPPS remains negatively charged in a large array of chemical environments, even under acidic pH and does not aggregate in media, allowing it to diffuse through cell walls and membranes more easily Issawi et al. 2019). Therefore, this PS was thought as an excellent candidate for APDT.
Under white light, TPPS at a very low concentration (MFC = 1.5 µM) induced a severe inhibition of B.
cinerea mycelium growth which led to death. We did not succeed to rescue the mycelium after this treatment. To our knowledge, there is little to no information available on the fungistatic or fungicidal effect of light-activated photosensitizers on B. cinerea (Imada et al 2014). The first step was to investigate whether the anionic porphyrin was able to induce any changes in the mycelium structure.
In the previous study, it was shown that the structure of the B. cinerea hyphae changed after treatment with antibiotics, eugenol, FeSO4 and tea tree oil (Šašek and Musilek. 1974;Wang et al. 2010;Fleurat-Lessard et al. 2011;Shao et al. 2013). We found that TPPS also induces a structural change on the fungus. More specifically, the structure of the PS-treated fungus was thinner than the structure of the control. The treated mycelium produced spores implying that the fungus was under stress. Further analyses, such as the H 2 O 2 content, MDA, MTT, total thiols and microscopy analysis, confirmed fungus stress to the point of dying. Our findings proved that TPPS, due to its characteristics, could be a valid alternative to classic fungicides ).
The grapevine varieties studied were Chardonnay, Merlot and Sauvignon. The choice of these three varieties was due to their different susceptibility to B. cinerea (Dubos 2002;Fermaud et al 2011).
Furthermore, as expected and according to our previous studies (Guillaumot et al. 2016;Issawi et al. 2018b), TPPS did not induce any phenotypical or biochemical modification of the three grapevine plantlets. In addition, this TPPS concentration (12.5 µM) was approximately ten times higher than the minimal concentration inhibiting the mycelium growth (1.5 µM); thus, the strategy presented in this article could work against pathogens without altering plant growth and development.
The final aim was to demonstrate TPPS efficiency against B. cinerea in two-month-old infected grapevine leaves to validate the hypothesis of mimicking a pathosystem. The in vitro cultures and artificial system indeed demonstrated the potential of our strategy. ESEM images from infected leaves, of the three grapevine varieties, after 72 h treatment confirmed that these leaves infected with B. cinerea pre-treated with 50 µM TPPS were similar to that of the control. For the Sauvignon variety, the presence of spores on infected leaves confirmed that it is more sensitive to the fungus infection than the other two varieties, which is also confirmed by the total thiol assay Conclusion TPPS was able to kill the pathogen B. cinerea without harming the grapevine leaves in vitro.
Moreover, this molecule does not produce any biochemical or phenotypical changes on the plantlets grown in vitro. These preliminary experiments carried out are indeed promising and, in the future, experiments could be done in a greenhouse and in fields to determine the real potential and efficacy of TPPS against plant pathogens. The results and findings presented herein are also very encouraging because the photodynamic treatment has been developed using a low concentration of PS. Therefore, we show that APDT can be used for the struggle against phytopathogens in the agronomic practices as the PS is effective against plant pathogens and exhibits non-toxic side effects toward plants.

Consent for publication
Not applicable.

Ethics approval and consent to participate
Not applicable.

Funding
The research is funded by the Région Nouvelle-Aquitaine.    Grapevine varieties growing on control medium and on medium supplemented with 12.5 µM TPPS. Pictures correspond to plantlets from each variety, cultured during one month in glass tubes containing control medium (left) or medium with 12.5 µM TPPS (right).

Figure 5
TPPS pre-treatment of B. cinerea leads to inhibition of mycelial growth on grapevine leaves.
Pictures were taken after a 72 hour infection of B. cinerea. Isolated leaves from the three varie-ties, without any contact with Botrytis (upper panel). On the three other panels, 4-dayold Botry-tis mycelium plugs were placed on isolated leaves. Before contact with isolated leaves, the plugs were pre-incubated or not with 12.5 or 50 µM TPPS for 8 hours under dark conditions. Subse-quent to pretreatment with 12.5 µM TPPS, mycelium growth was reduced, but infection was not inhibited. The lower panel corresponds to mycelium pretreatment with correspond to the 6 mm myceli-um disc placed on the leaf surface at the beginning of the experiments. Scale bar: 2 mm Figure 6 Scanning microscopy observations of leaves infected or not by B. cinerea pretreated or not with TPPS. After treatment with TPPS, the fungus was unable to infect the Chardonnay and Merlot leaves. For the Sauvignon variety, spores have been observed on leaf surface, even after TPPS pretreatment.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Agricultural And Food Sciences
Medicine

text:
Genome Sequence of Campylobacter fetus subsp. venerealis NW_ED23, Isolated from Bovine Sheath Wash

Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, which is mostly characterized by reproduction problems. The strain reported in this study was isolated from bull sheath wash in South Africa.

B ovine genital campylobacteriosis belongs to list B of the notifiable diseases of the International Office of Epizootics (OIE) (1). A recent study reported that the Campylobacter fetus subsp. venerealis bv. intermedius genome sequence is available, but the same study confirmed that more analyses were needed to verify if there are extrachromosomal replicons (2). Furthermore, van der Graaf-van Bloois and coauthors revealed that an unassembled genome does not allow full identification of the core and accessory genome and the reconstruction of pathways and surface structures that might contribute to the pathogenicity of this biovar (3). An increase in the prevalence of this biovar has been noticed in South Africa (4). Hence, there was a need to sequence this particular strain to determine if there is any deviation from that previously reported.
The sample used for the isolation of Campylobacter fetus subsp. venerealis NW_ED23 was collected in the North-West Province, South Africa, specifically, in Mamusa District Municipality, cultured on tryptose blood agar base CM0233 (Oxoid, United Kingdom) mixed with 7% sheep blood, and supplemented with Campylobacter selective supplement (Skirrow) SR0068E (Oxoid). The plates were incubated at 37°C for 72 h in a 2.5-liter anaerobic jar (Oxoid) with a CampyGen CN0025A sachet (Oxoid) to create microaerophilic conditions (5). The genomic DNA (gDNA) was extracted from a single colony using a Zymo Research kit (Merck, South Africa). The PCR was conducted using MG3F and MG4R primers for the amplification of the 16S rRNA gene (6). The Basic Local Alignment Search Tool (BLAST) of the NCBI website was used to confirm identity and similarity (7), and the accession number obtained was MT138648. The gDNA from the pure colony based on Sanger identification confirmation was used for library preparation using a NEBNext Ultra II FS kit according to the manufacturer's instructions and was used for the whole-genome sequencing.
The extracted gDNA sample was fragmented using an enzymatic approach (NEBNext Ultra II FS kit). The resulting DNA fragment was sized (200 to 500 bp) using AMPure XP beads. The fragment was end repaired, and Illumina-specific adapter sequences were ligated to each fragment. The sample was indexed, and a second size selection step was performed. The sample was then quantified using a fluorometric method and diluted to a standard concentration (4 nM). It was then sequenced on an Illumina NextSeq platform using a NEBNext Ultra II FS 300-cycle kit according to the manufacturer's instructions to generate a total of 516,191,294 reads with 2 ϫ 150-bp paired-end read length, and the coverage was 40ϫ.
The sequence data were analyzed with the KBase platform with default parameters (8). The sequenced data were filtered for low-quality reads and adapter regions using Trimmomatic v0.36 (9) and FastQC v0.11.5. The genome assembly was performed using SPAdes v3.13.0 (10). Functional annotation of the whole-genome sequence was performed with the NCBI Prokaryotic Genome Annotation Pipeline (11) and Rapid Annotations using Subsystems Technology (RAST) v0.11 (12). Campylobacter fetus subsp. venerealis NWU_ED23 has a total length of 1,880,748 bp assembled in 161 contigs with an average GϩC content of 33.05%. The sequenced data generated contain 2,004 coding genes. The N 50 value of the genome was 26,493 bp, while the L 50 value was 22. The entire genome assembly contains 36 RNAs and 194 subsystems.
Data availability. The whole-genome shotgun project of Campylobacter fetus subsp. venerealis NWU_ED23 has been deposited in DDBJ/ENA/GenBank under the accession number JACASH000000000. The version published in this paper is JACASH010000000. The raw reads were also submitted to the NCBI SRA under accession number SRX8607292, BioSample number SAMN15356083, and BioProject number PRJNA641553.

ACKNOWLEDGMENT
This project was funded by the North-West University through the Animal Health Department.


text:
Development of Pea Breeding Lines with Resistance to Orobanche crenata Derived from Pea Landraces and Wild Pisum spp.

Pea (Pisum sativum) is an important grain legume worldwide whose cultivation is severely constrained by the root parasitic weed crenate broomrape (Orobanche crenata), which is widespread in the Mediterranean Basin and Middle East. No resistance is available in commercialized cultivars but some levels of incomplete resistance has been reported in pea landraces and Pisum spp. relatives. In this paper we report the development of a number of advanced pea breeding lines with resistance derived from wide crosses with resistant P. fulvum, P. sativum ssp. elatius, P. sativum ssp. syriacum, and with pea landraces, and critically discuss current progress and future perspectives on pea breeding for broomrape resistance. Resistance of breeding lines was confirmed over five field trials, showing markedly reduced broomrape over ground emergence, and in rhizotron experiments, showing either reduced tubercle formation or, in some of the lines, also hampered tubercle development that might grow slower or even become necrotic and die. Breeding lines performed well agronomically, having similar or mostly higher yields than the parental pea cv. Messire in environments with high broomrape incidence.

Introduction
Pea (Pisum sativum L.) is a cool season legume grown worldwide as a source of protein both for human food and animal feed. Significant efforts have been made in pea breeding for disease resistance in continental and oceanic conditions where it is mainly spring sown [1]. Relative prevalence and importance of the various diseases varies with the agroecological conditions. The root parasitic weed broomrape (Orobanche crenata Forsk.) is widespread in the Mediterranean Basin and Middle East, and with climate change, it is spreading further north in Europe, and further south in Africa [2]. In this area broomrape is the major constraint for pea production [3,4]. So far, the effectiveness of conventional control methods is limited due to numerous factors, in particular the complex nature of the parasite, which reproduces by tiny and long-living seeds [5].
Breeding for broomrape resistance is difficult considering the scarce and complex nature of resistance in legumes in general [6] and in pea in particular. Only incomplete resistance to O. crenata was found in other grain legumes such as faba bean (Vicia faba L.), which has been successfully accumulated by breeding, allowing the release of resistant cultivars [6][7][8]. A similar effort on pea breeding for broomrape resistance has only recently been started with no resistance available in any pea cultivar or breeding line by that time [3], but various levels of resistance reported in germplasm accessions of P. sativum ssp. sativum, abyssinicum, arvense, and elatius, and in Pisum fulvum Sibth. & Sm. [9][10][11]. Resistance to O. crenata in pea has been reported to be quantitative and polygenic [12] and highly influenced by the environment, complicating the selection of the most resistant genotypes. Identified resistance to O. crenata in pea has been characterized at various levels from histology to proteomics [9,[12][13][14], but underlying gen(es) are still unknown. The rapid development of pea genomic tools will certainly facilitate characterization and use in breeding of the still undiscovered resistance genes. Meanwhile, we progressed in crossing and selection under field conditions and succeeded in development of a number of advanced breeding lines resistant to broomrape infection that are described here.

Materials and Methods
The pea network described here consisted of 9 pea breeding lines derived from the IAS-CSIC pea breeding program [11]. In brief, crosses were made among (partially) resistant accessions (P. sativum ssp. sativum Ps624 and Ps423, P. sativum ssp. elatius P675, P. sativum ssp. syriacum P665, and P. fulvum P660) [9][10][11] and pea cultivar Messire and others, and submitted to pedigree yearly selection from F 2 . The lines described here include selection from single crosses (GC number), backcrosses (BC number), or multiple crosses (GCC number). At this stage of the breeding program, the first priority in selection was broomrape resistance, with agronomic performance being second. Taking into consideration that progenies originated from wide crosses, fertility, growth habit and other yield-related traits were low in early generations. Basically, within the most broomrape-resistant progenies, the ones that were most fertile and had better agronomical potential were selected, discarding the rest. As a result, 9 resistant and productive breeding lines were selected after 6 seasons of yearly selection under heavy and uniform O. crenata soil infestation conditions. Their performance was further characterized in the field in Córdoba (Spain) during three consecutive seasons and at Escacena del Campo (Huelva, Spain) during two seasons, and under controlled conditions using mini-rhizotrons.
A randomized complete block design with three blocks was used in all field studies. Pea cv. Messire, frequently used in early crosses and highly susceptible to O. crenata, was included as a check in all experiments. Each replicate consisted of 1 m 2 plots consisting of 3 rows 1 m long, separated by 0.33 m, with 10 plants per row. Sowings were carried out between December and January, according to local practice. At the end of the crop cycle, the number of emerged O. crenata shoots per pea plant (Oc/pl, Table 1) was scored by counting the total number of pea plants and the total number of emerged O. crenata shoots per plot. At plant maturity the plots were harvested. Grain yield was assessed by weighing the seeds produced per plot and estimating the corresponding grain yield (kg/ha).
The in vitro studies were performed using mini-rhizotrons as earlier described [9,15]. Pea seeds were surface sterilized with 2% (w/v) NaOCl solution and 0.02% (v/v) Tween 20 for 5 min and then rinsed thoroughly with sterile distilled water and germinated for 4 days in 9 cm diameter Petri dishes with moistened filter papers at 23 • C conditions. Three pea seedlings were individually transferred to glass fiber filter paper (GFFP) (Whatman International Ltd., Maidstone, UK) sheets and placed over square Petri dishes (12 cm by 12 cm) filled with sterile perlite moistened with sterile distilled water. The Petri dishes were previously punctured on the top to allow pea stem develop outside of the dish. The seeds of O. crenata were surface sterilized by immersion in 0.5% (w/v) NaOCl and 0.02% (v/v) Tween 20 for 5 min, rinsed thoroughly with sterile distilled water, and spread separately over the GFFP sheets at a density of 50 seeds per cm 2 . The Petri dishes containing the pea-O. crenata co-cultivation system were sealed with parafilm, wrapped in aluminum foil, and stored vertically in a growth chamber (23/20 • C, 16/8 h day/night). The plants received Hoagland's nutrient solution modified at one-quarter strength once per week. O. crenata seeds located at a distance of 3 mm from the pea roots were inspected under a stereoscopic microscope (Leica S9i, Leica Microsystems GmbH, Wetzlar, Germany) to determine (i) the percent of contacted O. crenata radicles that successfully penetrated pea roots and formed a healthy tubercle at 30 days after infection (dai), (ii) the percent of total formed tubercles that became necrotic and died by 45 dai, and (iii) the developmental stage of O. crenata tubercles at 30 and 45 dai: T1: tubercle with a diameter <1 mm without crown roots, T2: tubercle with a diameter >1 mm without crown roots, T3: formation of crown roots, and T4: formation of shoot [9].

Results
The focus of the yearly selection was on O. crenata resistance. As a result, selected progenies showed reduced O. crenata infection levels compared to the Messire pea cultivar used as the control ( Table 1) Pea selections overyielded cv. Messire in the environments with high O. crenata infection (Cor07 and Cor08), however, yields were similar or even lower than Messire when O. crenata pressure was low (Cor09 and Esc09). All pea selections resembled parental Messire in flower color and plant morphology except GCC99-J17, which still had colored flowers from wild parents, and the aphylla type, a trait probably inherited from the Ballet parent. Breeding line GCC136-J24 also showed the aphylla type, conferred from the Radley parent.
Rhizotron studies confirmed the observed field resistance of the selected breeding lines ( Table 2). Over 71% of the O. crenata seedlings successfully penetrated the roots of Messire and formed a tubercle, which developed fast and healthy on Messire roots, being already in the spider stage with formation of crown roots (stage T3) by 30 dai and in the stage of shoot development (stage T4) by 45 dai, none of them being necrotic. The capacity of O. crenata radicles to penetrate the pea roots and form tubercles was significantly reduced in all breeding lines, being in the range of 0.5-34.7% (>2 × fold reduction compared to Messire). In addition to resistance reducing tubercle formation, some breeding lines exhibited additional resistance hampering tubercle development. Those tubercles formed on the roots of GC248-NS46, BC20-J13, BC20-J15, and GCC124-J19, which developed more slowly than on Messire roots. The necrosis of O. crenata tubercles formed on BC20-J10 and GCC124-J19 was visible at 45 dai (50 to 100%, respectively).

Discussion
Reduced levels of O. crenata infection were successfully transferred from landraces of P. sativum ssp. sativum (Ps624, Ps423), P. sativum ssp. syriacum (P665), P. sativum ssp. elatius (P675), and P. fulvum (P660) to the adapted breeding lines described here. The resistance achieved was not complete, but it was at the levels of the donors and of other breeding lines [9,10,15,16].
Resistance against root parasitic weeds is a multicomponent event, being the result of a battery of avoidance factors, resistance mechanisms acting at different levels of the infection process, or both [11,17,18]. Rhizotron experiments confirmed that the reduced O. crenata infection in the pea breeding lines observed in the field was not the result of escape or avoidance as reported in some legumes [18,19], but rather was due to true genetic resistance preventing O. crenata tubercle formation and development. True resistance in pea has also been reported acting before haustoria development by reducing the induction of O. crenata seed germination [9,20]. However, this was not studied here since the roots of the donors of resistance of the described breeding lines induced levels of O. crenata germination at the level of susceptible lines [15]. Pre-haustorial mechanisms of resistance preventing Orobanche tubercle formation and post-haustorial ones hampering development of formed tubercles have been described in pea and a range of crops [9,15,17,[20][21][22][23][24][25][26].
The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to allow marker-assisted selection with the hope that this speeds up the delivery of improved cultivars to the farmer. A previous study on a Recombinant Inbreed Line population derived from the cross P665 *Messire shows a quantitative inheritance governed by several QTLs [12]. These were of rather small effect that precluded the development of markers to be used in Marker Assisted Selection (MAS). However, by classical field selection we succeeded in selecting a breeding line (GCC99-J17) with resistance derived from this P665 together with Pe675, reinforcing the value of classical breeding that allowed us to achieve some relevant progress, even in absence of the most wanted availability of molecular markers. The situation was similar for faba bean, in which a number of studies reported quantitative inheritance governed by a number of QTLs explaining rather little phenotypic variation, and therefore not yet used in MAS [27], which has retarded but not prevented the efficient breeding and release of resistant cultivars [6,7]. No genetic studies have ever been performed to unravel the inheritance of the resistance of Ps624 or Pf660. Based on current information, we cannot conclude on their inheritance. However, the fact that we succeeded in selecting resistance from Ps624 in progenies derived from a single cross, and from P660 in progenies of backcrosses with Messire or multiple crosses with other elite cultivars, allows us to speculate that inheritance of O. crenata resistance in pea might not be as complex as earlier suggested [11,12], and that efficient field selection is feasible.
The key to the development of cultivars with long-lasting broomrape resistance is diversity [8]. A first strategy could be pyramiding more than one gene or by combining monogenic and polygenic resistance with the help of reliable molecular markers for efficient selection. It is widely acknowledged that complex resistance is likely to be more durable than monogenic resistance. However, the durability of resistance is not solely an issue of the number of genes, but also depends on the mechanism of action of the genes. Rather than pyramiding several genes acting at the same level, combining different resistance mechanisms providing multiple barriers not easily overcome by parasite changes may provide more durable outcomes. For instance, the "low germination induction" resistance, likely to be singly inherited [8,20], could be most relevant for providing a durable outcome, particularly when used in combination with other mechanisms, such as the ones reported in these new breeding lines.
The accelerated progress in the genomic and biotechnological research faced with pea, with a genome sequence recently released [28], will soon facilitate the understanding of crucial developmental mechanisms in both the parasite and the host and will speed gene discovery and the development of breeder-friendly molecular markers [8]. Unfortunately, less is yet understood of O. crenata genomics, with some relevant info on related species such as Phelipanche aegyptiaca [29] and O. cumana [30] that might help in understanding parasite virulence and host resistance mechanisms. This will facilitate gene discovery that will accelerate breeding through the development of markers for efficient MAS, as already done in sunflower breeding for O. cumana resistance [31,32], or allow genome editing. Unfortunately, this is not yet available for pea/broomrape. Meanwhile, we progressed in identifying a range of sources of resistance and in introducing them into an adapted pea background through sexual crossing and yearly field selection.
The result is the development of a number of advanced pea breeding lines resistant to broomrape infection that are described here and that are available on request to be used as parents in breeding programs elsewhere. Selections described here overyielded the parent pea cultivar Messire when the O. crenata infection was high, although yields could be lower in the absence of O. crenata pressure. However, standing ability should still be improved.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.