Eur J Plant Pathol DOI 10.1007/s10658-017-1235-4

Environmental effects on growth and sporulation of Fusarium spp. causing internal fruit rot in bell pepper M. Frans

&

R. Aerts & S. Van Laethem & J. Ceusters

Accepted: 19 April 2017 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2017

Abstract Internal fruit rot in bell pepper (Capsicum annuum L.) is mainly caused by members of the Fusarium lactis species complex (FLASC) and to a lesser extent by Fusarium oxysporum and Fusarium proliferatum. Despite the importance of the disease, there is hardly no information about growth, sporulation and germination dynamics of FLASC. In order to understand the dominance of FLASC as main pathogen of internal fruit rot, the effects of temperature (5 °C – 35 °C), water activity (aw 0.76–0.96), pH (pH 3 pH 9) and oxygen concentration (2.5% - 20%) on growth and sporulation of all three Fusarium species were compared. In addition, germination kinetics were also investigated. FLASC showed optimal mycelium growth and sporulation in the narrow range of 25 °C, while both other strains were also tolerant for higher temperatures to 30 °C. FLASC was also characterized by a broad pH optimum from pH 3–7 while F. oxysporum (pH 4–7) and F. proliferatum (pH 5–8) were more demanding concerning pH. In addition, optimal sporulation occurred in the acid region for FLASC (pH 3) whilst neutral and alkaline pH were more favourable for the other species. Germination kinetics revealed that FLASC did not benefit from an earlier and/ or faster germination process. A thorough understanding of the growth characteristics and dominance of FLASC M. Frans (*) : R. Aerts : S. Van Laethem : J. Ceusters Faculty of Engineering Technology, Department of Microbial and Molecular Systems (M2S), KU Leuven, Technology Campus Geel, Kleinhoefstraat 4, 2440 Geel, Belgium e-mail: [email protected]

as main pathogen for internal fruit rot is inevitable to develop sustainable control measures for the disease. Keywords Fusarium lactis species complex . Internal fruit rot . Bell pepper . Mycelium growth . Sporulation . Germination

Introduction Sweet pepper or bell pepper (Capsicum annuum L.) is a high cash value crop grown worldwide. In northern hemisphere regions such as the Netherlands, Belgium, UK, Israel and Canada nearly all bell peppers are grown in protected environments such as greenhouses (Jovicich et al. 2005; Lin and Saltveit 2012). Most of these high-quality greenhouse produced bell peppers are supplied to the fresh market. Since 2000, this high quality is threatened by an important disease known as internal fruit rot, which has been found in bell pepper production worldwide (Utkhede and Mathur 2004; Yang et al. 2009; Choi et al. 2010). More recently, it became clear that the disease is not only restricted to greenhouse grown bell peppers, but also occurs in open field cultures in the Mid-Atlantic region of the USA (Kline and Wyenandt 2014). Only in northern hemisphere regions, where internal fruit rot has been studied already to some extent, annual major yield losses up to 5% with seasonal peaks even up to 50% have been attributed to internal fruit rot (Hubert et al. 2003; Utkhede and Mathur 2003; Yang et al. 2010; Frans et al. 2016). In open field cultures of the Mid-Atlantic

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regions, internal fruit rot has been observed at different rates between 1% and 50% depending on the cultivar. Peaks were observed in heavy rainfall periods prior to harvest (Kline and Wyenandt 2014). Infection starts when spores of the pathogens deposit on the stigma of the flower via air or via insects such as pollinating bees or bumblebees (Kharbanda et al. 2006). Hyphal growth can be observed on the stigma 12 h after inoculation followed by penetration of the pathogen through the style into the ovary tissues 6 days after inoculation (DAI) (Yang et al. 2010). After the initial infection, the disease stays latent until the ripening stage. During this stage, the fungus starts to proliferate as whitish-grey mycelium on seeds, placenta and inner surface of the infected fruit. External symptoms such as sunken lesions only appear at later stages, when the fruits already progressed through the supply chain towards the consumers (Yang et al. 2009; 2010). Identification of samples collected in Canadian greenhouses revealed that the pathogens causing the internal fruit rot disease in bell pepper belongs to different members of Fusarium. Initially, F. subglutinans was reported as the causal agent (Utkhede and Mathur 2004), but based on more extensive studies and molecular analyses, Yang et al. (2009) identified most of these isolates collected in Canadian greenhouses as F. lactis. In the Netherlands, Hubert et al. (2003) collected samples in different greenhouses in 2002 and identified them as F. oxysporum, F. solani and for then an unknown species of Fusarium. Inoculation of flowers in 2003 with those samples delivered fruits with severe infections of internal fruit rot. Sustaining on molecular analyses, Van Poucke et al. (2012) confirmed most isolates collected in Belgian (82), Dutch (9), British (6) and Canadian (1) greenhouses belonged to members of the Fusarium lactis species complex (FLASC) (75%), but also isolates of F. oxysporum (14%) and F. proliferatum (9%) have been detected. Isolates of infected field bell peppers in USA have also been identified as F. lactis (Kline and Wyenandt 2014). Fusarium lactis has originally been reported as a contamination in milk by Pirotta and Riboni in 1879. Currently, only a few known diseases are linked with F. lactis. The fungus has been reported as the causal agent for endosepsis in cultivated figs (Michailides et al. 1996) and more recently as one of the pathogens causing gall formation on Swietenia (Soto-Plancarte et al. 2013). In 1998, Nirenberg and O’Donnell suggested to classify the fungus in the Gibberella fujikuroi species complex based on extensive morphological studies.

Fusarium oxysporum is the most widely dispersed and, without a doubt, the most economically important species of the Fusarium genus. Especially because of its numerous hosts and tremendous yield losses after infection (Leslie and Summerell 2006). In Capsicum L., wilt, stem and fruit rot are caused by members of F. oxysporum (Alfieri et al. 1984; Cha et al. 2007; Lomas-Cano et al. 2014). Fusarium proliferatum on the other hand, is well known for causing ear rot of maize (Marin et al. 1995; Velluti et al. 2000; Doohan et al. 2003). Also linked to F. proliferatum is the wilt disease in rice (Desjardins et al. 1997), asparagus (Elmer 1990) and palms (Armengol et al. 2005). Because of their importance, numerous studies have been conducted on the influence of environmental factors on its growth, sporulation and germination (Nelson et al. 1990; Marin et al. 1995; Brennan et al. 2003; Xu 2003; Palermo-Llamas et al. 2012). Only limited information about growth characteristics of F. lactis is available albeit. Van Poucke et al. (2012) identified FLASC as the predominating agent causing internal fruit rot in bell pepper. Control measures are still insufficient and limited to trials of chemical and biological agents aiming to prevent infection of the bell pepper flower (Utkhede and Mathur 2005). To obtain a deeper understanding of FLASC dominance as the main pathogen of internal fruit rot in bell pepper in greenhouses, this paper describes germination kinetics and the effects of temperature, water activity, pH and oxygen concentration on: (a) growth rates, and (b) sporulation of the three Fusarium species causing internal fruit rot. The results of reduced growth and sporulation at different ambient conditions are important to understand differences in pathogenicity and prevalence in culture conditions and as such can improve breeding and selection processes for new cultivars. In addition, the obtained information can help to investigate the possibilities of a sustainable control by influencing environmental parameters during plant growth and development or after harvest.

Materials and methods Fungal isolates and inoculation F. lactis sequence type 1 (MUCL 51511), F. oxysporum isolate PSKW 2 and F. proliferatum isolate VM 07 strains were isolated from infected bell peppers collected at the Sint-Katelijne-Waver Research Station for Vegetable Production (PSKW), Belgium, and identified in the Institute

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for Agricultural and Fisheries Research (ILVO) Merelbeke, Belgium. The isolates were stored at −80 °C in the fungal collection of the Laboratory of Sustainable Plant Production and Protection, University Leuven, TC Geel. To perform experiments, isolates were cultured on Potato Dextrose Agar (PDA, Biokar Diagnostics, Beauvais Cedex, France) at 20 °C for 14 days. For all experiments, a minimal medium (MM) was developed, based on an adapted Czapek-Dox recipe by Leslie and Summerell (2006) with a pH of 5.5 and a water activity (aw) of 0.96 unless otherwise stated. Using a cork borer, 8.5 mm mycelial agar disks cut from 2week-old Fusarium colonies were aseptically transferred to the centre of a 9 cm petri dish containing 25 ml of MM. Temperature study For assessing the effects of temperature on the daily radial growth, ten plates for each Fusarium species were incubated for 7 days at 5, 10, 15, 20, 25, 30 and 35 °C in an incubator equipped with 75Wm−2 cool white light (Philips, Eindhoven, the Netherlands) (16 h light/ 8 h dark) and 80% relative humidity. Starting at the second day after inoculation, colony size was measured daily, on two perpendicular axes for each plate (n = 10), using a digital calliper. Average sporulation in spores per millilitre was determined by flooding five different plates each with 10 ml of sterile distilled water and dislodging the spores using a spatula. Quantification of the spores was established by using a haemocytometer (Bürker Türk, Marienfield, LaudaKöningshofen, Germany). Water activity (aw) study For the water activity studies, the MM was modified with different concentrations of glycerol (Dallyn and Fox 1980) to values of 0.96, 0.93, 0.89, 0.85 and 0.76. The water activity was checked with a LabMaster Aw (Novasina AG, Zurich, Switzerland). Daily growth (n = 10) and sporulation after 7 days on five plates for each aw level (n = 5) were determined as described above. pH study In the pH studies, MM was modified with a citric acid buffer (0.1 M sodium citrate and 0.1 M HCl) to achieve pH values 2.0, 3.0 and 4.0. For pH values 5.0, 6.0 and 7.0

a phosphate buffer was used, whilst a boric acid – borax buffer was added to the MM to obtain pH values of 8.0 and 9.0 (Gomori 1955). The acidity was checked with a Polyplast BNC electrode (Hamilton, Bonaduz, Switzerland). Daily growth (n = 10) and sporulation after 7 days (n = 5) were determined as previously described. Oxygen study To study the effect of oxygen on the radial growth, inoculated plates were placed into 4 different conditions: Air (about 20% O2), 10% O2, 5% O2, 2.5% O2. For each condition, two plates of each Fusarium were packaged into five pouches (VAC090 PA/PE 20/70, Euralpack, Belgium) using a chamber machine (C 200, Multivac, Germany). After sealing, the pouches were incubated at 25 °C under cool white light (16 h light/ 8 h dark) and 80% RH for 4 days until O2 concentration dropped till 0.5%. Daily growth (n = 10) and sporulation after 4 days (n = 5) were determined as previously described. The concentration of O2 and CO2 were checked daily by piercing a needle trough a reusable septum. The needle was connected with a headspace analyser (Checkpoint O2/CO2, PBI Dansensor, UK). Spore germination The design used for spore germination was adapted from Sautour et al. (2001). In a 9 cm empty Petri dish, three sterile plastic cylinders (Ø 1.5 cm) were placed on the lid and filled with 250 μl of MM. After solidification of the medium, the cylinders were removed and the surface was ready for inoculation with 5 μL of 107 spores ml−1. After inoculation, 15 ml sterile water/ Tween 20 solution (0.01%) was poured in the Petri dish to preserve the relative humidity (100%). The Petri dishes were sealed with Parafilm® and incubated for 24 h at 25 °C (Fig. 1). This device allowed to observe the spores through the lid, without opening the dishes. By marking the dish, the germination of the same spores was observed every 2 h under a light microscope (Leica Microsystems, Wetzlar, Germany) at ×200 connected to an Olympus SC30 camera (Dantigny et al. 2005). Using a digital image analysis program (ImageJ, Schneider et al. 2012) germ-tube length for each spore was measured. When the length of the germ-tube was equal or longer than one half of the spore diameter, spores were considered germinated (Paul et al. 1992). For each

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Fusarium spp., 25 spores were observed per cylinder for a total of 10 cylinders for each Fusarium species (n = 10). Statistical analyses In all cases for mycelium growth, linear regression was applied to the increase in radius against time to obtain growth rates (mm/day) under each set of conditions. As growth followed a linear pattern between day 2 and 7 after inoculation, these points were selected as start and end point respectively for regression calculations (Excel 2003 Microsoft, Redmond, Washington, USA). Using SPSS (IBM© SPSS Statistics ver. 22, Armonk, NY) analysis of variance (ANOVA) followed by Tukey’s test was used to investigate significances of differences (α = 0.05) for all the treatments (i.e. temperature, water activity, pH and oxygen). In the germination assay, Vo50, which is the observed time required for 50% of the conidia to germinate, was calculated by linear interpolation from the data around 50%. The average observed rate of germination (roc) was obtained by linear regression between 4 and 14 h. Analysis of variance (ANOVA) followed by Tukey’s test was used to determine significance of differences (α = 0.05) for the three pathogens.

Results Temperature The optimum growth temperature for the three Fusarium isolates was 25 °C. Fusarium oxysporum showed a higher growth rate (15.42 ± 1.09 mm day−1) in comparison to FLASC and F. proliferatum with growth rates of 12.30 ± 0.65 and 10.92 ± 1.01 mm day−1 respectively. In contrast to F. oxysporum and F. proliferatum, which showed considerable growth in a range from 20 to 30 °C, growth rates of FLASC reduced by 50 and 70% for 20 °C and 30 °C respectively. At the low and high extremes, colony growth after 7 days was negligible for all fungi at 5 °C and 35 °C respectively. Sporulation patterns were similar as the growth patterns except at 30 °C for F. oxsporum and F. prolifertum. Highest spore production for FLASC (10.4 × 107 spores/ml ± 1.5 × 107) and F. oxysporum (12.8 × 107 spores/ml ± 1.9 × 107) was observed at 25 °C. Fusarium proliferatum on the other hand showed maximal sporulation at 30 °C (6.7 × 107 spores/ml ± 1.8 × 107) (Fig. 2).

Water activity (aw) The growth rate for the three isolates of Fusarium decreased significantly with decreasing aw. No growth was observed at or below a water activity of 0.85 and optimal growth was observed at the highest aw of 0.96 (Fig. 3). As expected, the highest spore concentration was found at aw 0.96 for FLASC (P < 0.05) compared to lower aw values. For F. oxysporum and F. proliferatum sporulation was not influenced by water activity concerning values of aw 0.96 and 0.93 (P > 0.05) (Fig. 3). pH With an optimal growth from pH 3 to 7, FLASC was less demanding concerning acidity in comparison to F. oxysporum and F. proliferatum with optimum growth at pH ranges of 4–7 and 5–6 respectively. At pH 3 growth was reduced by 50% for both F. proliferatum and F. oxysporum. F. proliferatum was most tolerant to alkaline conditions as growth had only slightly diminished at pH 8, whilst pH 9 inhibited growth for all Fusarium strains. The most obvious distinction between FLASC and the other Fusarium species is the optimal sporulation in the more acid region for FLASC, while a more neutral pH resulted in optimal sporulation for F. oxysporum and F. proliferatum. FLASC reached its highest sporulation at pH 3 (10.2 × 10 7 spores/ ml ± 1.0 × 107). Fusarium oxysporum (9.9 × 107 spores/ml ± 2.8 × 107) and F. proliferatum (3.7 × 107 spores/ml ± 0.7 × 107) reached an optimal sporulation at a more neutral pH of 7 (Fig. 4). Oxygen FLASC showed slightly reduced growth rates (about 15%) at oxygen concentrations lower than 20% whilst spore production was only significantly diminished (P < 0.05) at oxygen levels of 2.5%. On the contrary, F. oxysporum showed only significant slower growth rates (about 15%) at the lowest concentrations of oxygen but sporulation was not effected in these conditions. The sporulation was similar for F. proliferatum but its mycelium growth was equal (P > 0.05) for all the tested oxygen concentrations (Fig. 5). For all three Fusarium spp. spore production was lower than in the previous experiments due to the shorter duration of this experiment.

Eur J Plant Pathol Fig. 1 Example of the experimental device for observing Fusarium spp. spores microscopically without opening the dish as adapted from Sautour et al. 2001

Plastic cylinders

250 µl MM

Lid Petri dish

Parafilm®

15 ml sterile water/Tween 20

Spore germination Germination tubes were observed after 4 h for all three Fusarium species after inoculation and germination rates (roc) were similar for the three pathogens. However, a significant difference in the time when 50% of the viable spores had been germinated (Vo50) was noticed. Fusarium proliferatum reached Vo50 after 6.75 h ± 0.67 which was significantly faster (P < 0.05) than FLASC (9.12 h ± 0.78). Fusarium oxysporum on the other hand showed 50% germination of the viable spores after 8.12 h ± 1.75 (Fig. 6).

Discussion Over the years, internal fruit rot has emerged as a major worldwide problem in both greenhouse-grown bell peppers and open field cultures (Utkhede and Mathur 2004; Yang et al. 2009; Choi et al. 2010; Kline and Wyenandt 2014). Yang et al. (2010) found that the main pathogens causing this disease are members of Fusarium lactis and to a lesser extent Fusarium oxysporum and Fusarium proliferatum. Although internal fruit rot is a major threat

for bell pepper production, only limited information about FLASC has been published so far. In this study, the authors present objective data in order to investigate and comprehend the dominance of FLASC as the main pathogen causing internal fruit rot, despite having been reported previously as a weak pathogen (Yang et al. 2009). The optimum growth temperature for all three isolates was 25 °C. Sporulation was maximal at this temperature for F LASC and F. o xys poru m b ut F. proliferatum also showed a maximal sporulation at 30 °C. Using similar in vitro studies, other researchers have found various optimum growth temperatures. The same optimum temperature of 25 °C for mycelial growth have been reported for different F. oxysporum strains (Hibar et al. 2006; Scott et al. 2010; Webb et al. 2015) and for F. proliferatum (Palermo-Llamas et al. 2012). However, other reports showed an optimal growth at 30 °C for F. proliferatum on various growth media (Marin et al. 1999; Samapundo et al. 2005). To understand the effects of environmental conditions on the sporulation dynamics of Fusarium on aborted fruits, conidia were counted on all experiments. In the temperature experiment, spore production followed the same

Fig. 2 Plots of (a) colony growth rate (mm/day) (n = 10) and (b) sporulation (106 conidia ml−1) (n = 5) versus temperature (°C) for FLASC, F. oxysporum and F. proliferatum. Error bars represent standard deviations

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Fig. 3 Plots of (a) colony growth rate (mm/day) (n = 10) and (b) sporulation (106 conidia ml−1) (n = 5) versus water activity (aw) for FLASC, F. oxysporum and F. proliferatum. Error bars represent standard deviations

pattern as mycelial growth, giving maximal sporulation on the optimal growth conditions with the exception for F. prolifertum, which showed equal sporulation at 30 °C and 25 °C. Similar results observed in other Fusarium species can confirm its ability to develop in a wide range of temperatures (Marin et al. 1995; Doohan et al. 2003; Tonapi et al. 2007; Rossi et al. 2009). Several other studies demonstrate that temperature can have a significant effect on disease incidence (Bosland et al. 1988; Rossi et al. 2009). Temperature may not only affect pathogen growth, but also disease susceptibility of the host. Daily air temperatures in bell pepper greenhouses vary between 23 °C and 26 °C, while night air temperatures are around 21 °C. Higher temperatures during summer may influence the plant’s ability to constrain the pathogens’ development.

In general, Fusarium spp. are considered to be moderately sensitive to water stress with an optimum aw of 0.99 and a minimum of 0.89–0.93 aw (Griffin 1981). All investigated isolates in this study showed optimal growth at 0.96 aw. The decrease of aw had a significant effect (P < 0.05) on the mycelial growth rates of all three isolates. Water activity (aw) can also influence sporulation of Fusarium species. Yet in our experiments, optimum sporulation was observed at 0.96 aw. These results confirm similar findings by Marin et al. (1995) and Samapundo et al. (2005) for F. proliferatum. Highest growth rates for all isolates were found at pH 5 which is similar to an optimal pH reported by Marin et al. (1995) for Fusarium proliferatum. While most Fusarium species show poor tolerance on very acid pH levels (Wheeler et al. 1991), FLASC was able

Fig. 4 Plots of (a) colony growth rate (mm/day) (n = 10) and (b) sporulation (106 conidia ml−1) (n = 5) versus pH for FLASC, F. oxysporum and F. proliferatum. Error bars represent standard deviations

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Fig. 5 Plots of (a) colony growth rate (mm/day) (n = 10) and (b) sporulation (106 conidia ml−1) (n = 5) versus oxygen concentration (% O2) for FLASC, F. oxysporum and F. proliferatum. Error bars represent standard deviations

to tolerate pH levels of pH 3. Fusarium proliferatum on the other hand, grew well in more alkaline media. Fusarium oxysporum and F. proliferatum sporulation was optimal at pH 6–7 and pH 7–8 respectively, while FLASC reached highest sporulation pH 3. Together with its modest growth at low pH levels, this result may support the dominance of FLASC as main pathogen. As pH slightly decreases form pH 5.20– 5.93 in green bell peppers to an average pH of 4.65– 5.45 in red bell peppers, this might be a possible explanation for the dominance of FLASC as main pathogen causing internal fruit rot. Considering modified air packaging (MAP) as a possible solution to keep internal fruit rot in the latent phase, the effect of decreased oxygen levels on the mycelial growth of all three pathogens was also

Fig. 6 Plots for spore germination (%) (n = 10) in relation to time for FLASC, F. oxysporum and F. proliferatum on MM at 25 °C. Error bars represent standard deviations

investigated. Oxygen levels of 10% and lower did significantly reduce mycelium growth for both FLASC and F. oxysporum. Sporulation was only reduced at the lowest O2 concentration of 2.5%. Polderdijk et al. (1993) showed that O2 levels of 2–5% slows down ripening and respiration during transit and storage of red bell peppers thereby increasing shelf life. In addition, our results indicate that MAP can also constitute a potential way to diminish growth and sporulation of Fusarium during transit and storage of coloured bell peppers. Besides growth and sporulation, the pattern and timing of conidial germination was also investigated in detail. In the majority of the cases, conidial germination in Fusarium is a rather quick process that can take place in periods of 4–7 h (Palermo-Llamas et al. 2012).

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Yang et al. (2010) observed hyphal growth 12 h post inoculation on bell pepper flowers. In our in vitro studies, first germination tubes were observed 4 h after inoculation for all three pathogens and germination rates roc were also equal. However, the time to reach 50% germination of all viable spores (Vo50) was significantly longer for FLASC (9.12 h ± 0.78) compared to F. proliferatum (6.75 h ± 0.67). As such the dominance of FLASC as the main causing agent of internal fruit rot cannot be attributed to a faster germination time or rate. In conclusion, the current research has highlighted important environmental factors that influence growth and sporulation of FLASC as causal pathogen of internal fruit rot. FLASC showed optimal growth in a narrow range centred around 25 °C. Concerning common daily greenhouse temperatures from 23 to 26 °C these conditions are ideal for the fungal development. The good growth and sporulation on low pH levels are an important factor that could favour the presence of FLASC in ripening bell pepper fruits. The results of reduced growth and sporulation at low O2 levels are important with regard to implementing potential post-harvest control measures such as modified atmosphere packaging by keeping the fungus in its latent phase. Acknowledgements The work was funded by IWT (Agency for Innovation by Science and Technology, IWT-LA 135088). The authors would like to thank Kurt Heungens and Kris Van Poucke from the Institute for Agricultural and Fisheries Research (ILVO) for providing the Fusarium isolates. We would also like to thank Kristine Hauglum Holter, Ann Karin Bjørhus and Ana Catarina Aleixo Silva for their assistance in the lab.

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