Stepwise screening of candidate antagonists for biological control of Blumeria graminis f. sp. tritici

Biological Control 136 (2019) 104008

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Stepwise screening of candidate antagonists for biological control of Blumeria graminis f. sp. tritici

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Jürgen Köhla, , Helen Goossen-van de Geijna, Lia Groenenboom-de Haasa, Betty Henkena, Rüdiger Hauschildb,1, Ulrike Hilscherc, Carin Lombaers-van der Plasa, Trudy van den Boscha, Mariann Wikströmd a

Wageningen University & Research, Droevendaalsesteeg 1, 6700 AA Wageningen, The Netherlands GAB Consulting GmbH, Ottenbecker Damm 10, 21684 Stade, Germany c Bayer CropScience Biologics GmbH, Metkenberg 6, 23970 Wismar, Germany d Agro Plantarum, Kärrarpsvägen 410, 265 90 Åstorp, Sweden b

A R T I C LE I N FO

A B S T R A C T

Keywords: Antagonists Biological control Blumeria graminis f. sp. tritici Powdery mildew Risk assessment Screening criteria Tilletiospsis pallescens Wheat

Antagonists for the biological control of Blumeria graminis f. sp. tritici were selected using a stepwise screening approach. Fungal colonizers of powdery mildew pustules were isolated from leaves of cereals and other plant species. Spore production, cold tolerance, drought tolerance and UV-B resistance as important characteristics for application of biocontrol candidates in the phyllosphere were tested in in vitro assays and preliminary risk assessments were conducted. Amongst 850 tested isolates 58% belonged to various taxonomical groups of Cladosporium. Only 3% belonged to species that have been reported in literature as antagonistic to powdery mildews. The stepwise screening approach allowed to reduce the number of candidate antagonists using screening criteria that can be tested reliably and cost-effectively in in vitro assays and by data mining from initially 1237 isolates down to 143 candidate antagonists belonging to 42 taxonomical groups. The potential of these isolates to reduce conidia production of B. graminis f. sp. tritici. in wheat was assessed in bioassays on potted winter wheat plants under controlled conditions. A set of ten superior isolates was subsequently tested in a series of trials on potted spring wheat plants under open field conditions. Isolates Tilletiopsis pallescens BC0441 and T. pallescens BC0850 significantly reduced the number of powdery mildew pustules per flag leaf by 30 to 62%. Slopes of the regression lines fitted to data on number of powdery mildew pustules during time were significantly reduced by the antagonists indicating that the powdery mildew epidemics were slowed down. Treatments with T. pallescens BC0441 and T. pallescens BC0850 also reduced leaf coverage with powdery mildew pustules in a small-scale field trial in spring wheat.

1. Introduction The use of biological control increased in agriculture and horticulture during the last decade and at present more than 100 biological control agents have been registered for commercial use in Brazil, Canada, EU, Japan, New Zealand and USA (van Lenteren et al., 2018). There is an increasing demand for new biological control products by growers to allow more and more completed biological disease control systems. Needs for biological control products in European arable crops, vegetables crops and perennial crops have recently been identified by Lamichhane et al. (2017). In this study, powdery mildew pathogens in various crops were identified as economically important resulting in high ranking research priorities for new biological control

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options against powdery mildews. Powdery mildew diseases are caused by a broad range of obligate biotrophic plant pathogens, usually with a narrow host range. In cereals and several other Gramineae, Blumeria graminis causes powdery mildew symptoms on leaves, stems and ears. Different formae speciales infect different species, e.g. B. graminis f. sp. tritici is attacking wheat. This pathogen has a polycyclic epidemic with short cycles with conidial infections and formation and release of new conidia within a few days (Cao et al., 2012). Various fungi have been evaluated as antagonists of different powdery mildew pathogens (Hijwegen and Buchenauer, 1984; Hijwegen, 1989; Kiss, 2003). In the past, biocontrol research focused mainly on the species Amphelomyces quisqualis, Pseudozyma flocculosa,

Corresponding author. Present address: APIS Applied Insect Science GmbH, Kurze Straße 3, 21682 Stade, Germany.

https://doi.org/10.1016/j.biocontrol.2019.104008 Received 19 February 2019; Received in revised form 3 June 2019; Accepted 12 June 2019 Available online 13 June 2019 1049-9644/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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maximum of five samples per site were collected from different plants. Sampled leaves were fixed on a plastic grid (6 mm mesh) which was inserted into a 50 ml tube to protect the powdery mildew pustules during transportation and mailing. Samples were processed within one to three days.

Tilletiospsis spp. and Verticillium lecanii (Dik et al., 1998). Commercial biological control products have been registered in several countries for applications in grapevine and many vegetable crops based on A. quisqualis, effective against various species of Erysiphales, under the trade name AQ10 (Kiss et al., 2004), and for applications in roses and cucumber based on P. flocculosa under the trade name Sporodex (Paulitz and Bélanger, 2001). No biological control products are available for control of powdery mildew in cereals. Screening of new microorganisms for commercial use in biocontrol of plant pathogens is a complex process because many criteria have to be considered besides the antagonistic efficacy, ranging from ecological fitness needed for good field performance to potential risks for humans, non-target organisms or the environment, growth in fermenters for mass production, and aspects of legal property rights and marketing (Whitesides et al., 1994; Alabouvette et al., 2009; Köhl et al., 2011). A stepwise screening approach has been proposed in which various criteria are considered with a focus on assays with low costs per isolate during the initial steps and a focus on more complex and costly assays with few selected isolates during the final screening steps. The objective of the study was to collect candidate antagonists from powdery mildew infected leaves and to select fungal isolates which fulfil major criteria for use in biological control of powdery mildew caused by B. graminis f. sp. tritici in wheat crops. Screening steps proposed by Köhl et al. (2011) were applied. The targeted disease had been defined based on industrial marketing studies (Step 1) before the start of the study that focused on the choice of origin and isolation techniques (Step 2), rapid throughput screening experiments (Step 3), and data mining (Step 4). Rapid throughput screening included tests on first assessments of sporulation capacity, major ecological characteristics such as cold tolerance, drought tolerance and UV-B resistance as well as the common safety criterion that isolates should not be able to grow at human body temperature. Data base mining included risk assessments to exclude potential pathogens of humans, animals or plants and to exclude isolates inciting potential environmental risks. Furthermore, a search was done on possible restrictions in use because of existing patent protection for certain genera, species or strains. Selected isolates were tested for efficacy under controlled conditions (Step 5) and mass production (Steps 6 and 7), and first field testing (Steps 8) was done for a subgroup of selected antagonists. The different screening steps of this case study have also been analysed with the aim to organize and further optimize future screenings, to avoid pitfalls and to improve the power of certain screening steps.

2.2. Isolation of fungal colonizers of powdery mildew pustules Sterile tap water (0.5 ml) was added to the bottom of each tube containing a leaf sample to enhance humidity in the tube and leaf samples were incubated for three days at 18 °C in the dark. From two powdery mildew pustules of each leaf sample, clumps of powdery mildew conidia possibly colonized by other fungal species were transferred on oat meal agar (OA; oatmeal 20 g l−1; technical agar, 15 g l−1) and malt extract agar at 1/10 strength (1 g malt extract l−1, technical agar 15 g l−1), both media amended with 100 mg l−1 streptomycin and 15 mg l−1 tetracycline. Touching of green or necrotic leaf surfaces with the inoculation needle was avoided. Per sample, two plates per medium each with four inoculations points were prepared and plates were incubated for seven to 14 days at 18 °C in the dark. Subcultures from the developing colonies of hyphomycetes were prepared on OA with a maximum of three isolates (with different colony morphology) from the same leaf sample. Developing hyphal tips were sub-cultured again to obtain pure cultures. Fungal isolates were stored on potato dextrose agar (PDA; CM139, Oxoid; 39 g l−1) agar in slants at 5 °C and on TSC cryopreservation beads (Technical service consultants LTD) at −80 °C. 2.3. Pre-screening of fungal isolates Fungal isolates were incubated on OA at 18 °C for 21 days in the dark. Plates were flooded with 10 ml of tap water containing 0.01% Tween 80 and spores and hyphae were removed from the agar surface by gently rubbing with a sterile rubber spatula. The suspensions were filtrated through sterile nylon gauze with a mesh of 200 μm. The concentrations were determined with the aid of a haemocytometer and the spore production per agar plate was calculated. Isolates producing less than 1 × 105 spores per plate were discarded. For the remaining isolates, spore suspensions were prepared containing 1 × 104 spores ml−1 and kept on ice until use. Spore suspensions were plated in sterile wells (24 wells-plate format with a well diameter of 16 mm; 10 µl suspension per well) containing 1.5 ml malt agar (1 g malt extract, 15 g agar, 1,000 ml tap water). For each isolate one well per plate was inoculated. Different sets of plates with inoculated wells were incubated at 5 °C, 18 °C and 36 °C in the dark for 21 days. Another set of plates was incubated at 18 °C and exposed to UV-B (290–320 nm) (Philips narrowband TL20W/01-RS ultraviolet-B) during the first seven days for eight hours per day at a rate of 1.1 and 4.1 W m−2 followed by 14 day incubation in the dark. The UV-B rate under the lids of the petri dishes had been determined with a spectrometer (Jaz spectrometer, Ocean Optics). An additional set of plates with wells containing malt agar adjusted to approximately −7 MPa and −13 MPa by adding KCl (Campbell and Gardner 1971) was incubated at 18 °C for 21 days. Wells were inspected for fungal

2. Materials and methods 2.1. Sampling of leaves with powdery mildew pustules Leaf samples with powdery mildew pustules were collected in The Netherlands, Western, Central and North Eastern Germany, and Sweden from March to July 2014. Cereal fields and grasses were inspected for powdery mildew, most likely Blumeria graminis, but also other plant species, most likely infected by various other powdery mildew species, were also inspected in agricultural fields, forests, road sides and gardens. Coordinates, vegetation type and plant species were recorded. A

Table 1 Selection criteria set for ecological characteristics in the pre-screening of fungal isolates on agar media inoculated with spore suspensions. Characteristic

Basic criterion

Stronger criterion used for specific fungal species

Purity Sporulation Safety Cold tolerance Draught tolerance UV-B radiation

Regular colony development and conclusive ITS or EF1 DNA sequences Production of 1x105 spores per Petri dish on oat meal agar after 21 d at 18 °C No germination and growth at 36 °C on malt agar during 21 d Germination and growth at 5 °C on malt agar during 14 d Germination and growth at −7 MPa on malt agar at 18 °C during 14 d Germination and growth after exposure to 1.1 W m−2 on malt agar at 18 °C during 14 d

– – – Germination and growth at 5 °C on malt agar during 7 d Germination and growth at −13 MPa on malt agar at 18 °C during 14 d Germination and growth after exposure to 4.1 W m−2 on malt agar at 18 °C during 14 d

2

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removed so that pots with six plants with fully developed leaves showing limited growth during the period of the assay were used in bioassays. Fungi. 143 fungal isolates belonging to 42 different taxonomical groups were tested in bioassays. The candidate antagonists were grown for 21 days on oat meal agar (20 g oat meal, 15 g agar, 1,000 ml tap water) at 18 °C. Suspensions of spores or yeast cells were prepared by flooding cultures with sterile tap water containing 0.01% Tween 80, gently rubbing with a sterile rubber spatula and filtration through sterile nylon gauze with a mesh of 200 μm. Concentrations of the suspensions were determined with the aid of a haemocytometer and adjusted to 1 × 107 spores or cells ml−1 by adding sterile tap water containing 0.01% Tween 80. Blumeria graminis f. sp. tritici (Bgt) originating from FAL92315 (originally obtained from by Dr Patrick Schweizer, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany) was maintained on wheat plants cv. Julius in a growth cabinet with 16 hrs light per day. Temperature was 20 °C during day and 17 °C at night and relative humidity was RH = 75%. For inoculum production for bioassays, six pots each with seven 7-day-old wheat plants cv. Julius were placed beside diseased plants in the growth cabinet for seven days. Ten to 16 leaves with powdery mildew pustules were cut from these newly infected plants and used within 1 h for inoculation of plants in bioassays. Detached leaves or whole pots with abundant sporulation of Bgt produced in the same way were used for inoculation of open field trials 1 to 3 with spring wheat plants grown in pots. Treatments and incubation. Pots with six wheat plants were placed in a spray cabinet on a rotating disk and spray-inoculated until run-off with spore suspensions of the different antagonist candidates using sterile atomizers. Control plants were treated with tap water containing 0.01% Tween 80. There were six replicates (pots) per treatment except for the untreated control where two sets of six pots were used. Treated pots were arranged in a block design with complete randomization within the six blocks. After spray inoculation, pots belonging to the same block were placed into a high humidity chamber (sized 710 × 440 × 380 mm L × W × H) at 15 °C and 16 h light per day. After 24 h, pots belonging to the same experimental block were placed together in an inoculation box (sized 1150 × 980 × 880 mm L × W × H) for inoculation with Bgt. Ten to 16 leaves with sporulating Bgt were placed on a sieve on top of an opening on the upper side of the inoculation box. The sieve with the leaves was covered with a metal cylinder into which compressed air was blown for 20 s. Conidia were allowed to settle during 30 min and the number of conidia mm−2 was determined on haemocytometers which had been placed horizontally amongst the pots. If needed, the inoculation was repeated to achieve a density of approximately 10 conidia mm−2 on the haemocytometer. Assessments. Phenotyping and photosynthesis efficiency. The percentage of leaf surface covered with powdery mildew pustules and the photosynthesis efficiency was measured in each bioassay seven days after inoculation with Btg for plants belonging to blocks 1 to 3. Young leaves developed during the course of the bioassay were removed before the assessment. Measurements were done with the Pathoscreen system developed by Phenovation (Wageningen, The Netherlands). The attached leaves were placed horizontally on the measuring platform. Pots were turned before a second measurement so that leaves were measured on two opposite sides. The total leaf surface was measured by the area showing chlorophyll fluorescence and the leaf surface covered with pustules on the leaves was measured using a specific colour ratio setting for the pustules. The percentage powdery mildew coverage was calculated as the mean of the measurements of both leaf sides. Additionally, photosynthesis efficiency was determined during the first measurement (Vredenberg, 2008). Quantification of Bgt conidia production. In each bioassay, two destructive samplings of leaves were done to assess the number of conidia per leaf after an incubation period of 11 d.p.i. using plants from blocks 1 to 3 and 14 d.p.i. using plants from blocks 4 to 6. The oldest leaf per

growth after seven, 14 and 21 days and the ability of isolates to produce fungal colonies under the different conditions was recorded. Selection criteria set for the different characteristics are shown in Table 1. 2.4. Identification of fungi Spores or mycelium from fungal colonies grown on OA were used for isolation of genomic DNA. Fungal tissues were lyophilized and total DNA was extracted using Sbeadex mini plant kit (LGC) and KingFisher™ Flex (Thermo Scientific). Lyophilized fungal tissue was disrupted using the TissueLyser II (Qiagen) and one stainless steel bead (3.2 µm) for 30 sec with a frequency of 30 Hz. After disruption, 200 µl lysis solution with 0.5 µl RNase (2 mg ml−1) was added to each sample and further DNA extraction was according to the protocol supplied by the manufacturer. ITS1/ITS4 DNA amplification (White et al., 1990) and EF1728F/EF2 DNA amplification (Carbone and Kohn, 1999; O'Donnell et al., 1998) were performed. Quality and quantity of the ITS1/ITS4and EF1-728F/EF2 PCR products (4 µl) were checked by electrophoresis on 1.0% agarose gels, purified and sequenced by Macrogen Europe (Amsterdam, The Netherlands). DNA sequences were analyzed using blastn (http://www.ncbi.nlm.nih.gov/BLAST) with the default parameters. Identification of taxonomical groups generally was based on ITS1/ITS4 DNA sequences (% similarity was 99 to 100%). For differentiation within the genera Cladosporium and Fusarium EF1-728F/EF2 DNA sequences were used (% similarity was 99 to 100%). 2.5. Risk assessment The potential risks to humans, the environment (including groundwater) and non-target organisms were assessed for isolates which fulfilled the basic selection criteria regarding their ecological characteristics. Data requirements for microbial plant protection products and their active substances according to Regulation (EC) 1107/ 2009 (Anonymous, 2009) served as guidance. Published literature was searched for potential pathogenicity or infectivity of the species to humans or other non-target organisms, such as mammals, birds, fish, aquatic invertebrates, algae, terrestrial plants and arthropods. Similarly, the potential of the species to produce metabolites that might have a negative impact on humans, non-target organisms and the environment (including groundwater) was assessed. Using this information, potential risks for operators performing the actual research work can be considerably reduced. Only if reports on negative effects of the species and the metabolites it might produce were absent, strains were further developed as potential biological control agents. Any report on adverse effects led to a “No go” decision. If sequence information was not sufficient for a precise determination at species level, isolates were classified as ‘Could be critical until species is determined’. 2.6. Patent search Possible patent protection was assessed for fungal species (or genera) fulfilling the basic set of selection criteria and not being classified as pathogenic or toxic to humans, plants or animals, thus classified as ‘no go’ and excluded from the further screening process. The worldwide database Espacenet (http://nl.espacenet.com) was used to find any patents protecting the use of a fungal species in biological control of plant pathogens. 2.7. Bioassays with wheat seedlings Plants. Winter wheat plants cv. Julius were produced from seeds in pots (70 mm in diameter; seven seeds per pot) containing 250 ml standardized potting soil in a greenhouse with 16 hrs light per day and temperature set at 20 °C during day and 16 °C at night; RH was 60–70%. Ten-day-old plants were used in bioassays. The smallest plant and youngest not fully developed leaves of the remaining plants were 3

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one detached leaf per pot was fixed on a stick at flag leaf height. Small pots with two infested plants were fixed at flag leaf height in Trial 2 and small pots with seven infested plants were placed on the soil amongst the wheat cv. Calixo plants in Trial 3. Experimental set up of trial in spring wheat crop. An experiment in a spring wheat crop cv. Heron was done in summer 2016 in Wageningen, the Netherlands. The spring wheat crop was sown on 21 March 2016 in an organically managed field in sandy soil. No crop protection products were applied in the field during the growing season. Beginning of June, a natural powdery mildew epidemic developed on lower leaves. First pustules on young developing flag leaves were found on 16 June. Plots (sized 1 × 1 m) were established in the spring wheat crop with 1 m distance between plots. The untreated buffers between plots were also grown with spring wheat. The plots were located in five blocks (replicates) with four treatments arranged completely randomized within blocks. Plots were treated by spray applications using a knapsack sprayer as described above with (1) water containing 0.01% Tween 80, (2) C. delicatulum BC0707, (3) T. pallescens BC0441, and (4) T. pallescens BC0850. Spore concentration for all antagonist treatments was 2x107 spores ml−1 water containing 0.01% Tween 80. Spray applications were done on 9 June, 16 June and 23 June. Approximately 250 ml per plot were sprayed on the spring wheat crop until runoff. Assessments in open field trials with potted spring wheat. The number of powdery mildew pustules was determined for all flag leaves on one date (Trials 1 to 3) or on several dates (Trials 4 to 10). Only pustules visible from the top were counted. Additionally, twenty arbitrarily chosen flag leaves per pot were sampled in trials 10 on 7 July to determine the number of conidia of Bgt produced on flag leaves. The sampled flag leaves were pooled per plot and conidia were washed off by shaking for 15 min in 50 ml water containing 0.01% Tween 80. The concentration of the resulting conidial suspensions was determined microscopically using a haemocytometer. The surface of the flag leaves was measured using a Li-COR inc.LI-3100 AREA METER and the number of conidia of Bgt produced per cm2 flag leaf was calculated. The flag leaf of spring wheat cv. Calixo wheat from Trials 3 and 6 were assessed for possible symptoms of phytotoxicity and senescence. In Trial 3, five leaves per pot were cut on 10 June and subsequently incubated in moist chambers for 7 d at 15 °C with 16 hrs light per day. The percentage leaf surface with symptoms of yellowing, redness and necrosis was estimated. In Trial 6, leaves were cut on 22 June and processed in the same way. Assessments in trial in spring wheat crop. The number of powdery mildew pustules was determined for all flag leaves in the central square of each plot (sized 0.3 × 0.3 m). Pustules visible from the top were counted on 22 June, 24 June, 28 June, 30 June and 4 July. On 28 June, 30 June and 4 July, the percentage of coverage of flag leaves with powdery mildew pustules was estimated. Ten flag leaves per plot were sampled on 4 July outside the central part of plots which was reserved for disease assessments in the field. The sampled flag leaves were pooled per plot and the number of conidia produced per cm2 flag leaf was determined as described above.

plant was cut. The six cut leaves per pot were transferred carefully, minimizing losses of Bgt conidia during handling, into 50 ml tubes with 10 ml water containing Tween 80 at 0.01% and shaken on a vortex (Retsch) for 5 s. The concentration of conidia in the obtained suspension was determined using a haemocytometer and expressed as number of conidia per leaf. 2.8. Open field trials Fungal inocula. For assessments in open field trials, isolates of Aureobasidium pullulans and Cladosporium spp. were grown and spore suspension containing 1x107 or 2x107 conidia ml−1 were prepared as described for bioassays. C. delicatulum BC0707 conidia were also produced in solid state fermentation on a cereal based growth medium, separated from the growth medium and dry spores were stored at 5 °C. Such conidia were suspended in tap water containing 0.01% Tween 80 and applied in open field trials 8 to 10 with spring wheat grown in pots and in the experiment in a field grown spring wheat crop. Isolates of Tilletiopsis pallescens were grown in glucose yeast extract broth (25 g glucose, 10 g bactopepton, 1 g yeast extract, 1000 ml tap water) in 2 L conical flasks containing 800 ml of the broth. Flasks were inoculated with 3 ml of a suspension containing approximately 107 cells ml−1 produced for 14 d on oatmeal agar at 20 °C. Thereafter, flasks were incubated on a shaker at 16 °C for 6 d. Resulting suspensions were centrifuged with 15,000g for 10 min at 4 °C, pellets were resolved in tap water containing 0.01% Tween 80 and suspensions were filtered through nylon gauze with a mesh of 200 µm. The concentration of budded cells and frequently present mycelial fragments was determined using a haemocytometer and was adjusted to 1x107 or 2x107 spores (or mycelial fragments) ml−1 using tap water containing 0.01% Tween 80. Experimental set up of open field trials with potted spring wheat. Ten trials were carried out in Wageningen, the Netherlands, in spring 2016. Spring wheat cv. Calixo was produced in pots (12 L, containing ‘Lentse potgrond nr. 4’). Ten plants per pot were grown for 14 days in an unheated greenhouse and thereafter for approximately eight weeks in the open field but protected from rain under a roof. Plants were watered and fertilized when needed and no crop protection products were applied. The ten plants per pot developed on average 52 tillers with fully developed flag leaves when used for trials. In total, ten sets of pots were produced at weekly intervals. For each of the ten open field trials, pots were placed in the open field (not grown with a crop) at a distance of 1 m between pots, arranged in five blocks (replicates) with one pot for each treatment with spore suspensions of different antagonists and two pots treated with water containing 0.01% Tween 80 as control. Treatments were arranged completely randomized with each block. In each trial three spray applications at weekly intervals were done using a compressed air-driven knapsack sprayer with one nozzle (Birchmeier helico saphir 1.2, 2F-0.6) at 250 kPa until run-off, equal to an application rate of approximately 1200 L ha−1. During spraying, pots were surrounded by a shelter to protect neighbouring pots from drift. The ten subsequent trials started on 2 May (Trial 1), 10 May (Trial 2), 17 May (Trial 3), 24 May (Trial 4), 31 May (Trial 5), and 7 June (Trials 6 to 10). Trials 6 to 10 were located at different field sites with expected differences in microclimatic conditions and differences in natural Bgt inoculum load originating from surrounding wheat crops. In Trials 1 to 7 treatments were: (1) water containing 0.01% Tween 80; (2) Aureobasidium pullulans BC0827; (3) A. pullulans BC0828; (4) Cladosporium cladosporioides Clade 1 BC0612; (5) C. delicatulum BC0707; (6) C. delicatulum BC0714; (7) C. basiinflatum/cladosporioides BC0081; (8) Tilletiopsis pallescens BC0441; (9) T. pallescens BC0812; (10) T. pallescens BC0850; and (11) T. pallescens BC0902. In Trials 8 to 10, treatments were: (1) water containing 0.01% Tween 80; (2) A. pullulans BC0827; (3) C. delicatulum BC0707; (4) T. pallescens BC0441; and (5) T. pallescens BC0850. Trials 1 to 3 were artificially inoculated with B. graminis f. sp. tritici one day after the first application of antagonists using heavily infested wheat cv. Julius plants (see above). In Trial 1,

2.9. Statistics Data from bioassays on percentage leaf coverage with powdery mildew pustules 7 d.p.i., photosynthesis efficiency 7 d.p.i. and log10transformed data on the number of Bgt conidia per leaf 11 or 14 d.p.i. were analysed by analysis of variance (ANOVA) separately for each bioassay considering the block design with three replicates (blocks) per antagonist treatment and six replicates (two per block) in the watertreated control. Fishers protected LSD tests (p = 0.05; two-sided) were used to identify treatments which resulted in statistically significantly lower values compared to the control treatment only treated with Bgt. Data obtained in open field trials were analysed separately for each trial and each assessment date by ANOVA and effects of antagonist treatments were compared to the water-treated control using Fishers protected LSD-tests (p = 0.05; two-sided). Regression lines were fitted 4

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fungal genera (Supplementary Table 2). Cladosporium spp. were dominating with 495 isolates (58.2% of the isolates) followed by Phoma spp. with 82 isolates (9.6%), Aureobasidium spp. with 81 isolates (9.5%), Fusarium spp. with 30 isolates (3.5%), Alternaria spp. with 22 isolates (2.6%), Microdochium spp. with 17 isolates (2.0%), Tilletiopsis spp. with 13 isolates (1.5%), and Penicillium spp. with 12 isolates (1.4%), Acremonium spp. with eight isolates (0.9%), and Pseudozyma flocculosa with five isolates (0.6%). The remaining 85 isolates belonged to 48 different taxonomical groups, in many cases represented by only one or very few isolates. Amongst 850 tested isolates, several fungal groups were found which have been described as potential biocontrol agents against various powdery mildews by Kiss (2003), which were Acremonium strictum (seven isolates), Fusarium oxysporum (one isolate), Phoma glomerata (1 isolate), Pseudozyma spp. (five isolates), and Tilletiopsis spp. (13 isolates). Kiss (1993) listed also several Cladosporium spp. as being antagonistic to powdery mildews which cannot be directly linked to the almost 500 isolates of Cladosporium spp. representing 24 taxonomic groups tested in our study because of recent major revisions in Cladosporium taxonomy (Bensch et al., 2010, 2012). 3.2. Ecological characteristics In screening assays on agar using a 24 wells-plate format, spore suspensions of 844 isolates produced colonies at 18 °C within 7 days, and after 14 d all tested 850 isolates formed colonies. As an initial screening criterion for safe use of potential biocontrol agents, isolates were tested on agar for their ability for spore germination and mycelial growth on agar at 36 °C to exclude isolates which grow at this temperature. From the 850 tested isolates, almost 99% of the isolates did not produce colonies at 36 °C during 21 days and only 12 isolates formed colonises (Table 2). Isolates of Penicillium spp. tended to have more frequently the ability to grow at 36 °C compared to other fungal groups. The initial criterion for cold tolerance was defined as the ability of isolates for spore germination and mycelial growth on agar at 5 °C and to form colonies within 14 d under these conditions. 93% of the tested isolates fulfilled this criterion with differences between tested genera. Cladosporium spp. isolates fulfilled the criterion for 99% with no differences between the various taxonomic groups representing the genus Cladosporium (data not shown), whereas less than 80% of the tested isolates belonging to Alternaria, Microdochium, Penicillium or to the group of ‘remaining taxonomic groups’ and only two isolates out of eight isolates of Acremonium spp. grew at 5 °C within 14 days (Table 2). The ability to form colonies on agar at 5 °C within 7 days was also analysed as stronger selection criterion for cold tolerance. 50% of the tested isolates were able to form colonies under these conditions (Table 2). However, there was a striking differentiation between genera. 75% of the tested Cladoporium spp. isolates fulfilled this criterion whereas less than 20% of the isolates belonging to other genera or the group of remaining taxonomic groups formed colonies within seven days. Within the genus Cladosporium, 220 isolates (90.2%) out of 244 isolates belonging to taxonomic groups identified as C. bruhnei or C. bruhnei/C. allicinum were able to form colonies, whereas less than 62.0% isolates belonging to other taxonomic groups showed this ability (data not presented). Almost 95% of the tested isolates had the ability for spore germination and mycelial growth at −7 MPa within 14 d initially set as criterion for drought tolerance (Table 2). However, more than 20% of isolates from the genera Microdochium, Tilletiopsis and several taxonomic groups represented by few isolates (‘remaining taxonomic groups’) did not fulfil the criterion for drought tolerance. All isolates were also tested at −13 MPa. This stronger selection criterion resulted in more pronounced differentiation between genera. More than 95% of isolates belonging to Cladosporium or Penicillium formed colonies within 14 d, but only approximately 50% of the isolates belonging to

Fig. 1. Overview on screening steps and number of isolates tested.

to data from open field trials 8 to 10 on disease development during time for the individual plots and slopes of the regression lines were analysed by ANOVA followed by LSD-tests (p = 0.05; two-sided) for comparisons of the effects of antagonist treatments versus the watertreated control. 3. Results An overview on the screening steps and the number of isolates tested is shown in Fig. 1. 3.1. Isolate identification 1237 isolates of hyphomycetes were obtained from 504 leaf samples with powdery mildew pustules of which 387 isolates were not included in the pre-screening assays. In most cases, such isolates did not reach the minimum threshold for spore production set at 1 × 105 spores per agar plate. In some other cases, colonies showed irregular growth or DNA sequences did not allow conclusive identification, probably because impurities of the isolates. 850 isolates, representing 125 different taxonomical groups based on ITS1/ITS4 or EF1-728F/EF2 DNA sequences, were included in the pre-screening assays and were tested in vitro on agar for various ecological characteristics. The origin of the tested isolates is shown in Supplementary Table 1. Sixty seven per cent of the tested isolates had been isolated from powdery mildew pustules on leaves of cereals or grasses, thus most likely from various formae speciales of B. graminis. The remaining isolates were isolated from various other powdery mildew species of various herbal plants (29% of the isolates) and of trees, especially oak trees (4% of the isolates). The majority of the identified and tested isolates belonged to a few 5

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Table 2 Number of isolates belonging to various genera of hyphomycetes originating from powdery mildew pustules fulfilling screening criteria ‘no growth at 36 °C’, cold tolerance, drought tolerance and UV-B tolerance. Taxonomical group

Acremonium spp. Alternaria spp. Aureobasidium spp. Cladosporium spp. Fusarium spp. Microdochium spp. Penicillium spp. Phoma spp. Pseudozyma flocculosa Tilletiopsis spp. Remaining 48 OTUs Total 1

Number of total isolates

Number of isolates fulfilling selection criterion1 No growth at 36 °C

Cold tolerance

Drought tolerance

UV-B radiation

36 °C, 21 d

5 °C, 14 d

5 °C, 7 d

−7 MPa, 14 d

−13 MPa, 14 d

1.1 W m−2, 14 d

4.1 W m−2, 14 d

8 22 81 495 30 17 12 82 5

8 21 81 493 30 16 9 81 5

(100.0) (95.5) (100.0) (99.6) (100.0) (94.1) (75.0) (98.8) (100.0)

2 17 79 493 25 13 9 79 3

(25.0) (77.3) (97.5) (99.6) (83.3) (76.5) (75.0) (96.3) (60.0)

0 5 9 375 5 5 0 6 0

(0.0) (22.7) (11.1) (75.8) (16.7) (29.4) (0.0) (7.3) (0.0)

8 18 80 493 27 12 12 79 5

(100.0) (81.8) (98.8) (99.6) (90.0) (70.6) (100.0) (96.3) (100.0)

1 14 38 477 16 2 12 10 1

(12.5) (63.6) (46.9) (96.4) (53.3) (11.8) (100.0) (12.2) (20.0)

8 21 81 493 29 17 12 82 5

(100.0) (95.5) (100.0) (99.6) (96.7) (100.0) (100.0) (100.0) (100.0)

6 20 80 480 29 15 5 79 5

(75.0) (90.9) (98.8) (97.0) (96.7) (88.2) (41.7) (96.3) (100.0)

13 85 850

13 81 838

(100.0) (95.3) (98.6)

12 60 792

(92.3) (70.6) (93.2)

3 19 427

(23.1) (22.4) (50.2)

7 63 804

(53.8) (74.5) (94.6)

1 9 581

(7.7) (10.6) (68.4)

13 81 842

(100.0) (95.3) (99.1)

12 65 796

(92.3) (76.5) (93.6)

Percentage of tested isolates in brackets.

selection criteria. When the basic selection criteria were used in combination with the stronger selection criterion for drought tolerance ‘spore germination and growth at −13 MPa within 14 d’, more isolates were excluded and only 545 out of 850 isolates fulfilled this combination of criteria. The majority of isolates belonging to Cladosporium spp. fulfilled these combined criteria, and approximately 50% of the tested isolates belonging to Alternaria spp. Aureobasidium spp. or Fusarium spp. For other taxonomic groups, the majority of isolates was excluded. Using basic selection criteria in combination with the stronger criterion for cold tolerance ‘spore germination and growth at 5 °C within 7 d’, resulted in selection of 412 out of 850 isolates. Again, a high percentage of Cladosporium spp. isolates fulfilled the combination of criteria with 75%, whereas only 25% or less of the isolates belonging to other taxonomic groups fulfilled this combination of selection criteria. A combination of the basic selection criteria with the stronger selection criteria for drought tolerance and for cold tolerance resulted in the exclusion of almost all isolates except for isolates belonging to Cladosporium of which 75% of the isolates fulfilled this combination of criteria and Alternaria spp. of which 23% of the isolates fulfilled this combination of criteria. Within the genus Cladosporium, percentages of isolates fulfilling combined selection criteria were similar for the major taxonomical

Alternaria, Aureobasidium or Fusarium, and approximately 10% of the isolates belonging to Microdochium, Phoma, Pseudozyma, Tilletiopsis or to the group of ‘remaining taxonomic groups’. After a treatment with UV-B (290–320 nm) at 1.1 W m−2 for 8 h per day during seven days followed by 14 d incubation in the dark, 99% of the tested isolates were able to form colonies within 14 d (Table 2). After treatments at higher UV-B dose with 4.1 W m−2, 93% of the tested isolates formed colonies. Isolates belonging to Penicillium tended to show higher sensitivity to 4.1 W m−2 with only five out of 12 isolates being able to form colonies after the treatment. A combination of basic criteria for antagonists for application in the phyllosphere were used for isolate selection: ‘no growth at 36 °C’, ‘spore germination and growth at 5 °C within 14 d’, ‘spore germination and growth at −7 MPa within 14 d’, and ‘spore germination and growth after UV-B radiation at 4.1 W m−2’. The majority of the tested isolates with 730 out of 850 isolates fulfilled the combination of selection criteria (Table 3). More than 85% of the isolates belonging to the genera Aureobasidium, Cladosporium and Phoma fulfilled the combined criteria whereas from most other genera approximately 50% of the isolates were selected based on the combined criteria. Only for isolates belonging to Acremonium or Penicillium, the majority of isolates was excluded and less than 20% of the isolates fulfilled the combined basic

Table 3 Number of isolates belonging to various genera of hyphomycetes originating from powdery mildew pustules fulfilling combined screening criteria. Taxonomical group

Acremonium spp. Alternaria spp. Aureobasidium spp. Cladosporium spp. Fusarium spp. Microdochium spp. Penicillium spp. Phoma spp. Pseudozyma flocculosa Tilletiopsis spp. Remaining 48 OTUs Total

Number of total isolates

8 22 81 495 30 17 12 82 5 13 85 850

Number of isolates fulfilling combined selection criteria1 Basic criteria

Basic criteria, stronger criterion for drought tolerance2

Basic criteria, stronger criterion for cold tolerance3

Basic criteria, stronger criterion for drought and cold tolerance

1 15 78 478 24 7 2 72 3 7 43 730

0 12 38 463 13 1 2 8 0 1 7 545

0 5 9 367 5 2 0 6 0 3 15 412

0 5 5 356 3 1 0 4 0 0 4 378

(12.5) (68.2) (96.3) (96.6) (80.0) (41.2) (16.7) (87.8) (60.0) (53.8) (50.6) (85.9)

(0.0) (54.5) (46.9) (93.5) (43.3) (5.9) (16.7) (9.8) (0.0) (7.7) (8.2) (65.2)

(0.0) (22.7) (11.1) (74.1) (16.7) (11.8) (0.0) (7.3) (0.0) (23.1) (17.6) (48.5)

(0.0) (22.7) (6.2) (71.9) (10.0) (5.9) (0.0) (4.9) (0.0) (0.0) (4.7) (45.3)

1 Criteria were: No colony formation within 21 d at 36 °C, colony formation during 14 d after incubation at 5 °C, at −7 MPa, and after UV-B exposure at 4.1 W m−2. 2 Colony formation during 14 d at −13 MPa. 3 Colony formation during 7 d at 5 °C.

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Table 4 Number of isolates belonging to Cladosporium spp. originating from powdery mildew pustules fulfilling combined screening criteria. Taxonomical group

Cladosprium australiense C. australiense/C. phaenocomae C. basiinflatum/C. cladosporioides/C. ramotenellum C. bruhnei C. bruhnei/C. allicinum C. cf. cladosporioides 1 C. cf. cladosporioides 1/C. cf. cladosporioides 2 C. cf. cladosporioides 4 C. cladosporioides C. delicatulum C. exile C. exile/C. pseudocladosporioides C. inversicolor C. lycoperdinum C. phyllactiniicola C. phyllophilum C. pseudocladosporioides C. ramotenellum/C. basiinflatum/C. cladosporioides C. scabrellum C. sinuosum/C. herbarum Cladosporium spp. C. subtilissimum C. varians C. xylophilum Total

Number of total isolates

Number of isolates fulfilling combined selection criteria1 Basic criteria

Basic criteria, stronger criterion for drought tolerance2

Basic criteria, stronger criterion for cold tolerance3

Basic criteria, stronger criterion for drought and cold tolerance

6 8 8

6 8 5

(100.0) (100.0) (62.5)

6 8 5

(100.0) (100.0) (62.5)

4 4 4

(66.7) (50.0) (50.0)

4 4 4

(66.7) (50.0) (50.0)

181 62 25 1

173 62 24 1

(95.6) (100.0) (96.0) (100.0)

169 62 22 1

(93.4) (100.0) (88.0) (100.0)

157 55 10 0

(86.7) (88.7) (40.0) (0.0)

153 55 8 0

(84.5) (88.7) (32.0) (0.0)

32 4 27 1 1 32 5 1 11 7 6

32 4 27 1 1 31 5 1 10 7 6

(100.0) (100.0) (100.0) (100.0) (100.0) (96.9) (100.0) (100.0) (90.9) (100.0) (100.0)

30 4 26 1 1 30 5 1 10 6 6

(93.8) (100.0) (96.3) (100.0) (100.0) (93.8) (100.0) (100.0) (90.9) (85.7) (100.0)

24 3 20 0 0 22 2 1 2 5 2

(75.0) (75.0) (74.1) (0.0) (0.0) (68.8) (40.0) (100.0) (18.2) (71.4) (33.3)

22 3 19 0 0 22 2 1 2 5 2

(68.8) (75.0) (70.4) (0.0) (0.0) (68.8) (40.0) (100.0) (18.2) (71.4) (33.3)

4 1 64 2 2 4 495

4 1 61 2 2 4 478

100.0) 100.0 (95.3) (100.0) (100.0) (100.0) (96.6)

4 1 58 2 1 4 463

(100.0) (100.0) (90.6) (100.0) (50.0) (100.0) (93.5)

3 1 45 2 0 1 367

(75.0) (100.0) (70.3) (100.0) (0.0) (25.0) (74.1)

3 1 43 2 0 1 356

(75.0) (100.0) (67.2) (100.0) (0.0) (25.0) (71.9)

1 Criteria were: No colony formation within 21 d at 36 °C, colony formation during 14 d after incubation at 5 °C, at −7 MPa, and after UV-B exposure at 4.1 W m−2. 2 Colony formation during 14 d at −13 MPa. 3 Colony formation during 7 d at 5 °C.

3.4. Patent search

groups C. bruhnei, C. bruhnei/C. allicinum, C. cf. cladosporioides 4, C. delicatulum and C. inversicolor (Table 4). Isolates of C. cf. cladosporioides 1 and C. phyllophilum tended to show lower cold tolerance so that only 10 of 25 tested isolates of C. cf. cladosporioides 1 and only 2 of 11 tested isolates of C. phyllophilum fulfilled the basic criteria combined with the stronger selection criterion for cold tolerance. The percentages of isolates fulfilling the different combinations of selection criteria were similar for isolates from different regions (data not shown). Less isolates obtained from powdery mildews on tree leaves tended to fulfil the stronger criterion for cold tolerance (Supplementary Fig. 1A). Isolates collected in May to June tended to fulfil more often the basic selection compared to isolates selected in March or April (Supplementary Fig. 1B).

A patent search was done for fungal groups listed on Table 5 except for groups excluded after ‘no go’ decisions based on risks assessments. Patents on the use of fungal species for the biological control of plant pathogens were found for Acremonium alternatum (related to Acremonium strictum), Alternaria alternata, Aureobasidium pullulans, Cladosporium cladosporioides, Microdochium bolleyi, Penicillium sp., Penicillium vermiculatum, Penicillium frequentans, Phoma glomerata and Pseudozyma flocculosa (Table 6). The use of these species in biological control of plant pathogens is thus not novel. For Phoma glomerata, more specifically Phoma glomerata ATCC MYA-2373, the specific effect against powdery mildew and for Cladosporium cladosporioides isolate H39 a broad effect against plant pathogens has been claimed. It can be concluded that the use of newly isolated strains of all fungal species under study can potentially be used against powdery mildew without restriction by present patents. However, patent protection for use against powdery mildew is not likely for new isolates belonging to several species, especially Phoma glomerata, Cladosporium cladosporioides and Pseudozyma flocculosa (if the mode-of action is based on the patentprotected cellobiose lipid flocculosin).

3.3. Risk assessment The potential risks to humans, the environment (including groundwater) and non-target organisms were assessed for isolates which fulfilled the basic selection criteria regarding their ecological characteristics (Table 3). Only 11 taxonomic groups were classified as ‘Go’ and 26 as ‘No go’ (Table 5). For the majority of isolates representing 48 taxonomic groups the obtained sequence information was not sufficient for a precise determination at species level so that only the genus was known or available sequences were identical for more than one species. In this situation isolates were classified ‘Could be critical until species is determined’, in many cases with additional remarks (Table 5).

3.5. Bioassays with winter wheat seedlings Eight bioassays were conducted with 143 isolates belonging to 42 taxonomical groups. Isolates were randomly chosen for testing in individual bioassays, except some isolates, showing promising efficacy when tested first, that were tested twice or several times in independent assays. The inoculation with Bgt conidia resulted in a density of approximately 4.4 conidia mm−2 deposited on a horizontally glass slide 7

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Table 5 Assessment of potential risks of fungal taxonomical groups to humans, the environment (including ground water) and non-target organisms. Decision

Taxonomical group

Justification

‘Go’

Acremonium strictum, Embellisia abundans, Epicoccum nigrum, Helgardia sp., Microdochium phragmitis, Phoma glomerata, Phoma macrostoma, Pseudozyma flocculosa, Pyrenochaetopsis poae, Ramularia eucalypti, Ramularia vizellae fungal endophyte, Fusarium sp., Phoma sp., Phoma sp./Epicoccum sp., Phoma macrostoma/Epicoccum sp., Phoma macrostoma/P. herbarum, Tilletiopsis sp., uncultured fungus Alternaria infectoria/A. californica

No Information was found in the literature on toxicity, infectivity and pathogenicity to non-target organisms including mammals and plants, or on production of potentially harmful microbial metabolites.

‘Could be critical until species is determined’

Alternaria sp. Aureobasidium pullulans, Aureobasidium pullulans/A. proteae all Cladosporium spp. (see Table 4) Colletotrichum sp. Didymella sp., Microdochium sp., Mycosphaerella sp., Neosetphoma samarorum/Leptospaeria sp., Neosetphoma sp./Leptospaeria sp., Pleosporales sp., Ramularia sp., Septoria sp., Sporisorium sp., Stagonosporopsis sp. Penicillium sp.

‘No go’

Truncatella angustata Ascochyta sp., Botrytis cinerea/Sclerotinia sclerotiorum, Microdochium nivale, Mycosphaerella punctiformis, Phaeosphaeria sp., Phoma exigua var. exigua, Phoma exiqua/P. multirostrata, Plectosphaerella cucumerina, Plectosphaerella cucumerina/Colletotrichum pisi Colletotrichum acutatum/C. fioriniae Didymella exitalis Fusarium avenaceum, Fusarium avenaceum/F. tricinctum, Fusarium avenaceum/Fusarium arthrosporioides, Fusarium culmorum, Fusarium culmorum/F. venenatum, Fusarium lateritium, Fusarium lateritium/ Fusarium acuminatum, Fusarium lateritium/Fusarium torulosum, Fusarium sambucinum, Fusarium sporotrichioides, Fusarium tricinctum/F. acuminatum Penicillium expansum Phoma herbarum

amongst the pots during inoculation in the first assay. In assays 2 to 8 higher densities with 8.2 to 11.8 (average 10.3) conidia mm−2 were obtained. In the first assay, a low coverage with powdery mildew with 2.1% was observed 7 d.p.i. and few conidia were produced on Bgt treated control plants which had not been treated with candidate antagonists (Supplementary Table 3). For the other seven assays, the mean coverage with powdery mildew for water-treated plants was 12.8% (range 7.0 to 20.3%). The mean number of conidia produced per leaf 11 d.p.i. (backtransformed values) for all bioassays was 1.01 × 106 with a range for different assays from 0.47 × 106 to 1.77 × 106 and was 1.59 × 106 (range 0.89 × 106 to 2.44 × 106) 14 d.p.i. On control plants which had not been inoculated with Bgt, only few pustules of powdery mildew were observed. The mean (backtransformed) number of Bgt conidia per leaf for water-treated plants was 1.35 × 104 (range 0.31 × 104 to 3.74 × 104) 11 d.p.i. and 4.56 × 104 (range 1.86 × 104

Insufficient taxonomical information

Described as rare dematiaceous human pathogen, moreover metabolites isolated from Alternaria infectoria species-group could be potential phytotoxins Some species are pathogenic for plants, isolated metabolites are mycotoxins and/or have cytotoxic effects on mammalian cells Opportunistic/new human pathogen, but two A. pullulans strain approved under 1107/2009 Cladosporium includes plant pathogens, but also saprophytes. Some of them are known as sensitizers or mycotoxin producers A few species are known to be pathogenic to human Some species are plant pathogens

Crop and plant pathogens, some species are pathogenic to human, mycotoxin producers, but also strains without detrimental effects Associated with vascular plants, either as endophyte or as pathogen Plant pathogens

Plant pathogen, animal infection described Plant pathogen and related to asthma Known as general plant pathogen, especially cereal pathogen, produce several mycotoxins which may pose a risk for consumers of plants or processed products

Plant pathogen and mycotoxin producer Pathogenicity towards fish has to be assessed

to 14.79 × 104) 14 d.p.i., indicating a low level of potential interference between pots with different treatments during the duration of assays. Conidiation productivity defined as Log [Number of Bgt conidia produced 14 d.p.i. divided by the percentage of leaf coverage by powdery mildew 7 d.p.i.] was similar for water-treated plants for most bioassays. In bioassays 2 and 3 with the highest coverage of leaf surface with powdery mildew lower conidiation productivities were observed compared to the other bioassays. Results obtained with the various treatments with candidate antagonists in the eight bioassays are summarized in Fig. 2 and in Supplementary Fig. 2. The number of Bgt conidia produced per leaf during 11 d.p.i. was statistically significantly reduced by several candidate antagonists in six bioassays (Supplementary Fig. 2). Such antagonistic isolates belonged to Aureobasidium pullulans (BC0995), Cladosporium sp. (BC0436), C. cf. cladosporioides 1 (BC0612), C. inversicolor

Table 6 List of patents on the use of fungal species in biological control of plant diseases. Fungal species listed in Table 5 except species with ‘No go’ decision were assessed. Fungal species

Patent reference

Subject

Acremonium strictum Alternaria sp. Aureobasidium pullulans Cladosporium cladosporioides Microdochium sp. Penicillium sp.

CN103897992 (A) − 2014–07-02 US2009257984 (A1) − 2009–10-15 US7601346 (B1) − 2009–10-13 US2013164320 (A1) − 2013–06-27 EP0279676 (A2) − 1988–08-24 CN102344891 (A) − 2012–02-08 RU2322490 (C1) − 2008–04-20 KR20000036322 (A) − 2000–07-05 US6544512 (B1) − 2003–04-08 US2006105986 (A1) − 2006–05-18

Acremonium alternatum against verticillium wilt Alternaria alternata against Plasmopara viticola Aureobasidium pullulans against Fusarium Head Blight Cladosporium cladosporioides against plant pathogens Microdochium bolleyi against take all Penicillium against Ustilaginoidea virens Penicillium vermiculatum against various fungal pathogens Penicillium frequentans against various fungal pathogens Phoma glomerata against powdery mildew Formation of antimicrobial flocculosin

Phoma glomerata Pseudozyma flocculosa

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Fig. 2. Effect of various fungal candidate antagonists (blue dots) on leaf coverage with powdery mildew (PM) and conidiation productivity of B. graminis f. sp. tritici 14 d.p.i. in comparison to an untreated control (red dots). Conidiation productivity has been defined as Log [Number of Bgt conidia produced 14 d.p.i. divided by the percentage of leaf coverage by powdery mildew 7 d.p.i.]. Results of 8 bioassays on winter wheat cv. Julius. Means of three replicates (antagonist treatments) and six replicates (water-treated control). Blue lines separate treatments with values statistically different from the water-treated control (two-sided Fishers protected LSD tests; p = 0.05). Note: Axes scales may differ between figures. Table 7 Efficacy of fungal isolates significantly reducing coverage with powdery mildew (%), number of Bgt conidia produced 11 d.p.i. or 14 d.p.i. or Bgt conidiation efficiency in bioassays on winter wheat cv. Julius. Efficacy (%) in reducinga Isolate

Number of Bgt conidia per leaf 11 d.p.i.

Number of Bgt conidia per leaf 14 d.p.i.

Leaf coverage with PM pustules (%)

Conidiation efficacy 14 d.p.i.

Aureobasidium pullulans BC0827 A. pullulans BC0828 A. pullulans BC0995 Cladosporium sp. BC0201 Cladosporium sp. BC0436 Cladosporium sp. BC0991 C. basiinflatum/C.c./C r. BC0081 C. bruhnei BC0183 C. bruhnei/C. allicinum BC0540 C. bruhnei/C. allicinum BC0607 C. cf. cladosporioides 1 BC0155 C. cf. cladosporioides 1 BC0612 C. cladosporioides BC0851 C. delicatulum BC0505 C. delicatulum BC0707 C. delicatulum BC0714 C. inversicolor BC0894 C. xylophilum BC0872 Phaeosphaeria sp. BC0357 Tilletiopsis pallescens BC0441 T. pallescens BC0812 T. pallescens BC0850 T. pallescens BC0902 T. pallescens BC0904

7.1 −12.6/18.3 31.3*/17.0 −7.9 39.0*/−50.2 −14.0 4.6/4.5 5.5 −14.2 −20.5 −54.6 33.7*/26.0 18.0 5.9/0.9 44.0*/26.9 3.4/22.4 33.1/39.6* 0.0/31.3* −0.5 34.0*/15.0* 47.6* −40.6/68.8*/31.1/29.3 41.3*/39.9* 26.5/28.1/7.2

39.7* 39.4*/27.0 −6.0/27.5 27.1* 12.7/19.3 8.3 33.02*/11.2 26.0 34.5* 35.6* 14.0 4.1/17.6 9.4 27.8*/10.2 19.5/21.1 31.7*/13.9 41.8*/21.7 32.5*/11.0 32.1* 50.3*/8.0 60.5* 57.8*/89.1*/45.3*/70.7* 56.4*/46.4* 75.3*/71.5*/20.3*

3.9 −17.8/18.6b 30.3/2.2 −43.9 −1.6/–33.1 −50.4 −1.1/16.4 −37.5 41.0 21.4 −84.6 −26.5/−7.7 −10.0 25.6/4.7 38.7/–23.4 16.2/28.9 −16.3/34.8 −36.6/15.2 6.9 23.1/−59.7 13.6 7.3/86.3*/34.5/75.0* 20.6/25.2 38.8/56.7*/−4.4

29.5 47.4* −44.2 31.6 18.2/41.0 41.0* 37.6/−24.2 47.2* −11.4 −21.1 55.3* 23.8/17.6 48.8* 8.6/8.2 –32.4/38.6 8.8/−19.7 51.1*/−15.1 44.7*/−13.8 26.4 36.8/79.3* 56.0* 55.8/7.1/20.0/−35.2 39.7/8.6 60.3*/31.1/64.6*

a Original parameter values analysed separately per bioassay; asterics (*) indicates significant reduction (%) compared to control treatment. Results presented per bioassasy for isolates tested repeatedly.

with the exception of T. pallescens BC0902 which significantly reduced conidiation of Bgt in two repeated experiments. Conidia production by Bgt measured 14 d.p.i. was significantly reduced in seven out of eight bioassays by at least one candidate

(BC0894), C. xylophilum (BC0872) and T. pallescens (BC0441, BC0850, BC0902) (Table 7). These isolates reduced Bgt conidiation by 30 to 70%. The treatment effects were not reproducible in most cases for antagonists which have been tested twice in independent bioassays 9

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pustules by 30 to 62% at the end of the trials. Trial 9 started with a higher initial level of powdery mildew with 0.15 pustules per flag leaf in the untreated control compared to 0.07 and 0.00 pustules per flag leaf in Trials 8 and 10. No significant treatment effects of the two T. pallescens isolates were found. However, in plots treated with C. delicatulum BC0707 significantly more pustules were found compared to the untreated control. This effect may be due to inhomogeneity at the experimental site leading to higher powdery mildew incidence already on 7 June before the spring wheat had been treated for the first time with a powdery mildew incidence ranging from 0.15 to 0.20 pustules per flag leaf in control and plots later treated with A. pullulans BC0827 or T. pallescens isolates but an incidence of 0.29 pustules per flag leaf in spring wheat later treated with C. delicatulum BC0707. Regression lines were fitted to data on numbers of powdery mildew pustules during time for the individual replicate pots. The adjusted R2 for the various regression lines generally ranged between 80 and 99% and was below 80% only in a few cases. Slopes of the regression lines for treatments with T. pallescens BC0441 and T. pallescens BC0850 were significantly lower in Trials 8 and 10 confirming that the increase of the powdery mildew symptoms was slower after treatments with these antagonists compared to the control treatment and treatments with A. pullulans BC0827 or C. delicatulum BC0707 (Table 9; Fig. 3). In Trial 9, a significantly larger slope was found for the treatment with C. delicatulum BC0707 compared to the other treatments. The number of Bgt conidia produced on flag leaves was quantified on 7 July in Trial 10. Treatments with T. pallescens BC0441 and T. pallescens BC0850 reduced the number of Bgt conidia significantly by approximately 50% compared to the control treatment from 26 conidia cm−2 leaf surface to 11 and 14 conidia cm−2 leaf surface (backtransformed values), respectively. Possible phytotoxicity of treatments was assessed in Trials 3 and 6 on leaves treated in the field that were subsequently incubated in moist chambers. No significantly enhanced yellowing or necrosis was observed.

antagonist. Significant reductions by 20 to 90% were found for A. pullulans (BC0827, BC0828), Cladosporium sp. (BC0201), C. basiinflatum (BC0081), C. bruhnei/C. allicinum (BC0540, BC0607), C. delicatulum (BC0505, BC0714), C. inversicolor (BC0894), C. xylophilum (BC0872), Phaeosphaeria sp. (BC0357), and T. pallescens (BC0441, BC0812, BC0850, BC0902, BC0904) (Supplementary Fig. 2, Table 7). Results were reproducible in two independent experiments for T. pallescencs isolates BC0441 and BC0850. Generally, there were no correlations between measurements of conidiation 11 d.p.i.and 14 d.p.i. (quantified on a second set of pots in the same experiment) (Supplementary Fig. 2). Measurements of leaf coverage with powdery mildew also showed a high variability between different treatments. Mean values for candidate antagonist treated plants often exceeded values of water-treated plants. Significant treatment effects of candidate antagonists resulting in a reduction of powdery mildew were detected only in bioassay 4 and bioassay 7 for Tilletiopsis pallescens isolates BC0850 and BC0904 (Fig. 2; Table 7). Calculation of the conidiation productivity of Bgt, been defined as Log [Number of Bgt conidia produced 14 d.p.i. divided by the percentage of leaf coverage by powdery mildew 7 d.p.i.], gave additional information on possible efficiency of the tested isolates. There was a general trend that productivity of conidiation was negatively correlated with the leaf surface covered by powdery mildew (Fig. 2). Leaves treated with A. pullulans (BC0828), Cladosporium sp. (BC0991), C. basiinflatum (BC0183), C. cf. cladosporioides 1 (BC0155), C. cladosporioides (BC0851), C. inversicolor (BC0894), C. xylophilum (BC0872) and T. pallescens (BC0441, BC0812, BC0904) produced significantly less Bgt conidia per surface area covered by powdery mildew (Table 7). Photosynthesis efficiency was lowest in bioassays 1 and 2 and more variable in bioassays 3 to 8 (data not shown). No significant effects of applications of candidate antagonists on photosynthesis efficiency were found. The 143 isolates tested in bioassays belong to 42 different taxonomical groups of which 24 taxonomical groups belong to the genus Cladosporium (Table 8). Isolates which significantly reduced conidia production of Bgt 14 d.p.i. in at least experiment belonged to eight taxonomical groups, including six taxonomical groups of the genus Cladosporium. T. pallescens, represented by five individual isolates, showed the highest efficacy in reduction of Bgt.

3.7. Trial in spring wheat crop The organically managed spring wheat crop cv. Heron had fully developed flag leafs at the beginning of the trial on 9 June. Powdery mildew pustules were visible on lower leaves with an incidence of less than one pustule per plant. No pustules were found on flag leaves on 9 June when the first of three spray application of antagonists C. delicatulum BC0707, T. pallescens BC0441 and T. pallescens BC0850 were carried out followed by applications on 16 June and 23 June. The powdery mildew epidemic progressed in the crop and first pustules were observed on flag leaves on 16 June. On 22 June, 5.8 pustules per flag leaves were counted in water-treated control plots (Fig. 4 A). In C. delicatulum-treated plots, the number of pustules was higher compared to the control treatment with 7.5 pustules per flag leaf whereas the number of pustules in T. pallescens-treated plots was significantly lower compared to the control treatment with 4.8 pustules per flag leaf for T. pallescens BC0441 and 2.8 pustules per flag leaf for T. pallescens BC0850. During the following 12 days, the number of pustules decreased in all plots, possibly, because young colonies were washed off during rainfalls or because individual pustules grew in size and merged so that they could not be counted separately. In control plots and C. delicatulum-treated plots, 4.6 and 5.0 pustules per flag leaf were counted on 4 July, on T. pallescens-treated flag leaves significantly less pustules were counted with 3.2 pustules per flag leaf for T. pallescens BC0441 and 2.3 pustules per flag leaf for T. pallescens BC0850. The coverage of the flag leaf surface with powdery mildew was first estimated on 28 June. Flag leaves of water-treated control plots and C. delicatulum-treated plots were covered by powdery mildew for 5.8 and 5.0% (Fig. 4 B). Flag leaves in T. pallescens-treated plots had a significantly lower coverage with 3.4% for BC0441 and 2.8% for BC0850. The coverage with powdery mildew increased until the last assessment on 4 July. Treatment effects of both T. pallescens isolates were

3.6. Open field trials with potted spring wheat No symptoms of powdery mildew were found in potted spring wheat cv. Calixo before 7 June even in experiments which had been artificially inoculated with Bgt. After 7 June, powdery mildew developed in the various sets of potted spring wheat plants used for pot Trials 1 to 10. Only few pustules of powdery mildew were observed on flag leaves in Trials 1 to 7 (data not shown). Sets of plants used for these experiments had been sown from 22 February onwards at weekly interval and such plants already started to senesce beginning of June. In a few cases, statistically significant treatment effects on the number of pustules per flag leaf were found for individual assessment dates of these trials. In such cases, powdery mildew was slightly reduced by A. pullulans BC0827 (Trial 6), A. pullulans BC0828 (Trials 3 and 6), C. delicatulum BC0707 (Trial 4), T. pallescens BC0812 (Trials 3, 4, 6 and 7), T. pallescens BC0850 (Trials 3, 4 and 6) and T. pallescens BC0902 (Trials 3 and 7). In a few other cases, a statistically significant increase of powdery mildew was found for treatments with A. pullulans BC0827 (Trial 7), C. basi./clado. BC0081 (Trial 7), C. cladosporioides 1 BC0612 (Trial 4, 7), C. delicatulum BC0707 (Trial 7) and T. pallescens BC0441 (Trial 4 and 7). In Trials 8 to 10, an increase of the number of powdery mildew pustules was observed after 7 June during the following two to four weeks until onset of senescence of the flag leaves (Fig. 3). Treatments with T. pallescens BC0441 and T. pallescens BC0850 on 7, 14 and 21 June significantly reduced the number of powdery mildew pustules per flag leaf in Trials 8 and 10 resulting in a reduction of the number of 10

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Table 8 Efficacy of fungal isolates belonging to various taxonomical groups in reducing the number of conidia of Blumeria graminis f. sp. tritici produced 14 d.p.i. in bioassays with winter wheat cv. Julius. Taxonomical group

Number of isolates

Mean (range)a

Acremonium strictum Aureobasidium pullulans A. pullulans/A. proteae Cladosporium australiense C. australiense/C. phaenocomae C. australiense/C. phaeno. strain CPC 18,221 C. basiinflatum/C. cladosp./C. ramot. C. basiinflatum/C. cladosporioides C. bruhnei C. bruhnei/C. allicinum C. cf. cladosporioides 1 C. cf. cladosporioides 1/C. cf. cladosporioides 2 C. cf. cladosporioides 4 C. cladosporioides C. delicatulum C. exile C. exile/C. pseudocladosporioides C. inversicolor C. lycoperdinum C. phyllactiniicola C. pseudocladosporioides C. scabrellum C. sinuosum/C. herbarum C. subtilissimum C. varians C. xylophilum Cladosporium sp. Colletotrichum acutatum/C. fioriniae Helgardia ap. Microdochium phragmitis Microdochium sp. Neosetphoma samarorum/Leptospaeria sp. Penicillium sp. Phaeosphaeria sp. Phoma glomerata Phoma macrostoma/Epicoccum sp. Phoma macrostoma/P. herbarum Phoma sp./Epicoccum sp. Pseudozyma flocculosa Ramularia sp. Stagonosporopsis sp. Tilletiopsis pallescens

1 7 (2)b 1 3 2 1 4 (2) 1 18 8 9 (1) 1 9 5 9 (4) 1 1 9 (3) 2 1 8 2 1 2 1 3 (1) 8 (1) 1 1 1 2 1 1 1 1 3 (2) 2 1 3 (1) 1 1 5 (4)

−19.5 18.6 −6.1 −7.9 6.4 3.4 20.0 11.2 10.7 13.0 11.0 10.6 1.4 6.0 12.1 15.2 10.5 11.1 −5.9 −8.0 6.0 14.5 6.7 −12.4 −1.9 14.9 7.2 21.0 −0.9 3.9 16.5 27.7 18.9 32.1 −12.7 3.8 2.2 0.5 10.9 10.6 5.9 52.5

a b

(-5.5 to 39.7) (-20.9 to 2.1) (1.0 to 11.8) (11.9 to 28.5) (–23.2 to 36.8) (-8.3 to 35.6) (-11.9 to 20.9) (-7.8 to 26.1) (-5.2 to 15.3) (-5.3 to 27.5)

(-26.6 to 36.9) (-10.0 to −1.9) (-15.2 to 24.2) (5.3 to 23.7) (-16.8 to −8.0) (10.9 to 21.8) (–33.4 to 27.1)

(14.6 to 18.4)

(0.6 to 8.6) (-5.3 to 9.6) (-3.3 to 22.4)

(29.1 to 65.7)

For isolates. Number of isolates tested twice in brackets. Individual isolates of T. pallescens each tested 2 to 4 times.

increase the chance of detecting powdery mildew-specific antagonists adapted to the ecological niche. Isolating from symptom-free leaf surfaces as an alternative route was not employed because this may result in a majority of isolates belonging to common and abundant phyllosphere inhabitants not antagonistic to powdery mildews which may even outcompete powdery mildew antagonists in the pathogen-free environment. More than 1200 isolates of hyphomycetes different from powdery mildew fungi were obtained from powdery mildew pustules from a total of 504 assessed leaf samples, indicating that hyphomycetes generally are present in powdery mildew pustules. Such hyphomycetes may be present as ungerminated spores with mildews simply functioning as spore traps or may actively grow within the pustules. Part of such colonizers may hyperparasitize powdery mildew fungi (Hijwegen and Buchenauer, 1984). It was not the intention of the design of the study to analyse the fungal biodiversity and characteristics of powdery mildew colonizing isolates in relation to powdery mildew species, host plants, sampling sites or sampling dates. Therefore, the obtained information on fungal diversity within powdery mildew pustules is incomplete. 31% of the obtained isolates had been excluded from further analysis because they showed slow growth and sporulation on artificial agar media and were thus considered as not suitable for industrial mass production. Furthermore, isolation technique and media may have been selective

significant compared to the water-treated control. The increase of leaf coverage with powdery mildew tended to be slower in T. pallescenstreated plots compared to water-treated plot and C. delicatulum-treated plots. However, due to considerable variation between plots and low number of data points during time, regression lines fitted to data per plot did not all fit well and possible treatment effects on slopes of regression lines could not be analysed (data not presented). Flag leaves were sampled on 4 July in all plots outside central parts of plots, that were used for on-site assessments of powdery mildew pustules. The number of Bgt conidia was 525 conidia per cm2 in watertreated plots (data not presented). In T. pallescens-treated plots the number of conidia tended to be reduced by 32.3% for isolate BC0441 and by 18.8% for isolate BC0850. In many cases, conidia obtained from T. pallescens-treated plots showed a different appearance, often with symptoms of disintegration of the cytoplasm. The germability of such conidia has not been determined. 4. Discussion Isolates were collected from powdery mildew pustules from leaves with low disease severity incubated under moist conditions for three days to enhance the development of possible antagonists. Powdery mildew pustules as a source of potential antagonists were chosen to 11

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Fig. 4. Effect of treatments with spore suspensions of Cladosporium delicatulum BC0707, Tilletiopsis pallescens BC0441 and T. pallescens BC0850 on Blumeria graminis f. sp. tritici on spring wheat cv. Heron. A. Numbers of pustules cm−2 flag leaf surface. B. Coverage (%) of flag leaves with powdery mildew pustules. Bars indicate standard error of the mean (n = 5). Asterisks (*) indicates values per assessment date significantly lower than values for the untreated control (two-sided Fishers protected LSD tests; p = 0.05).

and no information on the diversity within individual pustules was analysed. The diversity of the assessed 850 isolates was high with 125 taxonomic groups. However, only a few dominating genera were found with Cladosporium, Phoma, Aureobasidium, Fusarium, Alternaria and Microdochium which together represent 86% of the isolates. Interestingly, only 3% of the identified isolates belonged to Acremonium strictum, Fusarium oxysporum, Phoma glomerata, Pseudozyma spp. or Tilletiopsis spp., species known as powdery mildew antagonists (Kiss, 2003), whereas representatives of Ampelomyces quisqualis or Verticillium lecanii, also commonly known antagonists of powdery mildews (Hijwegen, 1989; Askary et al., 1998; Kiss, 2003) were not found. Performance of antagonists applied for control of leaf or fruit diseases is often limited by low temperatures, low humidity and UV irradiation (Köhl and Molhoek, 2001; Jijakli and Lahlali, 2016). Improvement of formulations is seen as inevitable to protect such antagonists from adverse climatic stresses. In the study, selection steps were used to overcome such limitations by selecting antagonists able to germinate and grow at 5 °C, at −7 MPa and after UV-B irradiation. Interestingly, more than 90% of the 850 tested isolates fulfilled such individual criteria and almost 90% of the isolates combined these characteristics. It can be assumed that tested isolates, all originating from leaves were adapted to the typical environmental stresses in the phyllosphere. These results demonstrate the importance of the choice of the appropriate niche for isolating candidate antagonists. In the study, three antagonistic isolates of Trichoderma harzianum and three isolates of Clonostachys rosea from the collection of the institute had been included for

Fig. 3. Effect of treatments with spore suspensions of Aureobasidium pullulans BC0827, Cladosporium delicatulum BC0707, Tilletiopsis pallescens BC0441 and T. pallescens BC0850 on the number of pustules per flag leaf of potted spring wheat cv. Calixo exposed to open field conditions. Plants were treated on 7 June, 14 June and 21 June. A. Trial 8. B. Trial 9. C. Trial 10. Bars indicate standard error of the mean (n = 10 for control; n = 5 for other treatments). Asterisks (*) indicates values per assessment date significantly lower than values for the untreated control (two-sided Fishers protected LSD tests; p = 0.05). Table 9 Effect of Aureobasidium pullulans BC0827, Cladosporium delicatulum BC0707, Tilletiopsis pallescens BC0441 and T. pallescens BC0850 on slopes of regression lines fitted to the number of pustules per flag leaf of potted spring wheat cv. Calixo exposed to open field conditions. Mean number of pustules and assessment dates: see Fig. 3 A, B and C. Treatment

Trial 8

Trial 9

Trial 10

Control A. pullulans BC0827 C. delicatulum BC0707 T. pallescens BC0441 T. pallescens BC0850

0.183 0.215 0.142 0.121*1 0.083*

0.137 0.147 0.242* 0.145 0.087

0.165 0.158 0.143 0.067* 0.103*

1

Significantly different from control (LSD-test; α = 0.05).

12

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criteria for cold tolerance (germination and growth within 7 days at 5 °C), drought tolerance (germination and growth at −13 MPa within 14 days) and UV-B resistance (germination and growth after UV-B radiation of 4.1 W m−2 within 14 days). Those isolates were grown on agar to re-assess the number of produced spores per plate and superior producers were selected. For fungal groups represented only by few isolates, the combination of basic selection criteria was used and only isolates were excluded showing weak sporulation in the second round of sporulation tests. The execution of the bioassays for antagonist screening against Bgt on winter wheat seedlings demanded substantial resources since inoculum production of the biotrophic pathogen and the high number of candidate antagonists, production of wheat plants and the establishment and assessment of the assays is labour-intensive and also depends on adequate climate chambers. Screening assays showed variation in disease development between and also within individual experiments. Different disease parameters such as leaf coverage with powdery mildew pustules, and the number of conidia produced per leaf 11 or 14 d.p.i. showed no consistent correlations. Despite these difficulties typical for biocontrol trials with a biotrophic pathogen, sufficiently high disease pressures were achieved in the different assays so that the majority of isolates showing no or moderate antagonism could be excluded. The overall results of the series of bioassays allowed the successful selection of few superior groups of antagonistic fungi. Various fungal species have been described as potential antagonists of powdery mildew species (Kiss, 2003). Amongst the 42 taxonomical groups tested in the bioassays against Bgt on winter wheat several taxonomical groups or closely related groups with reported antagonistic potential were included. Hijwegen (1989) found that Acremonium strictum and Penillium sp. are antagonistic to Sphaerotheca fuliginea on cucumber. Several Cladosporium species have been described by Mathur and Mukerji, 1981) and Minuto et al. (1991) as antagonists of several different powdery mildew species. Phoma glomerata is a potential antagonist of powdery mildew of Plantanus occidentalis (Sullivan and White, 2000). Pseudozyma flocculosa is antagonistic to powdery mildew in roses (Traquair et al., 1988; Jarvis et al., 2007; Mimee et al., 2009). Antagonism of Tilletiopsis spp. including T. pallescens against powdery mildew species has been reported for barley (Knudsen and Skou, 1993), cucumber (Hijwegen, 1992; Urquhart et al., 1994; Urquhart and Punja 1997, 2002), roses (Ng et al., 1997), mango (Wafaa et al., 2012) and grapevine (Haggag et al., 2014). From the 42 taxonomical groups tested in the bioassays on winter wheat, isolates of A. pullulans, Cladosporium spp. and T. pallescens showed antagonism against Bgt. The few tested isolates belonging to Acremonium strictum, Pseudozyma flocculosa and Phoma glomerata did not affect powdery mildew in the bioassays on winter wheat. Based on the overall results, ten out of 143 isolates were selected for further testing in small-scale field trials. The series of 10 small-scale experiments with potted spring wheat was conducted in the period between 2 May and 21 June (first and last spray) to determine the potential of the applied antagonists under different climatic conditions favouring powdery mildew development. The overall results showed that powdery mildew did not develop during the early season before 7 June although potted plants had been artificially inoculated with the pathogen. After 7 June, epidemics developed in all sets of potted spring wheat and in neighbouring spring wheat fields. This was most likely due to a natural development of the powdery mildew epidemic not supported by artificial inoculations. Disease levels were reached that allowed the reliable quantification of treatment effects. Overall results of trials with potted spring wheat showed that isolates of T. pallescens have a potential to reduce powdery mildew on wheat. This was shown occasionally during trials early in the season when disease levels were low, but more consistent in trials in June when higher disease levels allowed a better estimation of treatment effects.

comparison (data not presented). None of these isolates fulfilled the combination of the ecological selection criteria. Isolates of these genera, generally very common in soil and on plant residues, were not common on powdery mildew pustules in the study. Amongst the 850 tested isolates, only one isolate of these genera, belonging of T. rossicum, had been found. Isolates not being able to grow at human body temperature are preferred in biological control for safety reasons. Surprisingly, the study showed that 99% of the 850 tested hyphomycetes originating from phyllospheres of various host plants were not able to germinate and grow at 36 °C. Only isolates of Penicillium spp. differed from this common pattern with 25% of the tested isolates being able to germinate and grow at 36 °C. ITS1/ITS4 and EF1-728F/EF2 sequence information was used for identification of the fungal isolates. This allowed the differentiation between 125 taxonomic groups and identification of 67 groups at species level. Identification at species level is necessary for the assessment of possible risks with the aim to exclude potential pathogens of humans, animals and plants and isolates with potential risks for the environment from further screening steps. For the remaining 57 taxonomic groups identification was possible at genus level or in some cases the obtained sequence information was similar for more than one species. In these cases risk assessments were very preliminary and have to be done again after additional sequence information will allow identification at species level. For 11 species no risks were identified in the preliminary risk assessment based on available data in literature. Twenty seven taxonomic groups were excluded because literature indicated plant pathogenicity, mycotoxin production or, in one case, fish pathogenicity. For the large group of isolates belonging to the genus Cladosporium risk assessments were not conclusive, also because significant changes in Cladosporium taxonomy (Bensch et al., 2010, 2012) make it difficult to link published information on Cladosporium species to representatives of newly described species or formae specialis. The workflow for the initial screening tests was efficiently organized. Spore suspensions were prepared from cultures grown on agar and the spore concentration was determined as a first indication for mass production. From the same cultures, samples of mycelium or spores were taken, stored at −80 °C and used later for DNA extraction and sequence-based identification. Only suspensions of isolates with spore production above the set minimum level were plated by pipetting on standard malt agar or on agar adjusted to low water potentials using a 24 wells-plate format. Plates were subsequently incubated at different temperature or UV-B conditions and assessed after incubation. This was usually done in sets of 40 candidate isolates tested together with several standard isolates serving as checks for reproducibility of the individual assays. Surprisingly high numbers of tested isolates fulfilled the basic criteria for cold tolerance, drought tolerance and UV-B resistance which had been considered as strong selection criteria at the beginning of the study. Although less discriminating than expected, these assays are needed to confirm these essential characteristics of isolates before testing them in more complex screening steps. Published information was analysed for identified species regarding potential risks and patent searches were done to check for possible restriction in use. The analysed sequence information of two gene regions was not powerful to identify all fungal isolates. In future studies more gene regions should be included for fungal DNA barcoding (Stielow et al., 2015) to obtain detailed taxonomical information for more isolates and thus allowing preliminary risk studies for more isolates. The number of isolates which passed the high throughput screening (Köhl et al., 2011; Step 3) and the data mining (Step 4) was considerably high. Because of resource limitations the number of isolates had to be restricted to approximately 140 isolates for the next screening step, in which the antagonistic potential of candidate isolates was assessed on winter wheat seedlings inoculated with Blumeria graminis f. sp. tritici (Step 5). To reduce the number of isolates, major fungal groups with large numbers of representatives were selected based on stronger 13

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In a preliminary small-scale field experiment, applications of T. pallescens isolates BC0441 and BC0850 significantly reduced the number of powdery mildew pustules on flag leaves and the leaf coverage with powdery mildew. Treatments with both isolates also tended to reduce the number of produced conidia that in many cases showed symptoms of disintegrated cytoplasm. Such symptoms were not observed for conidia obtained from water-treated plots. Different to the situation in bioassays conducted under controlled conditions with artificial inoculation of Bgt 24 h after antagonist application, the open field trials were carried out with plants already showing first pustules at the start of the experiment. In this situation antagonists may suppress conidia production in newly formed pustules as assessed in the bioassays. Additionally, suppression of conidiation in already existing pustules may also occur, limiting pathogen spread to healthy leaf parts. The measured treatment effects indicate that both antagonists have potential to control powdery mildew epidemics under field conditions. Under the conditions of the field experiment, three applications of non-formulated cells were carried out without knowledge on optimum timing and optimum concentration of antagonist applications. Small plots of spring wheat have been sprayed and observed for treatment effects that were located within a commercially grown spring wheat crop with a naturally occurring powdery mildew epidemic without any application of crop protection products. Under such experimental conditions, a constant influx of Bgt conidia from the surrounding infested crop into the experimental plots may have masked for a part the treatment effect of reduced numbers of conidia with possibly also reduced germability. As a consequence, treatment effects on the progression of the polycyclic disease probably may be underestimated. Tilletiopsis spp. are basidiomycetes with yeast-like growth, forming ballistospores, mycelium, budded cells (blastospores) and chlamydospores (Nyland, 1950; Boekhout, 2011). Information on the potential of Tilletiopsis spp. for biological control of powdery mildew is limited to few reports on preliminary work in cucumber, roses, mango and barley. Hijwegen (1992) reported on the antagonism of T. minor against powdery mildew in cucumber. Knudsen and Skou (1993) found that T. albescens caused a collapse of Erysiphe graminis f. sp. hordei on barley leaves and Sphaerotheca fuliginea (Fr.) Poll. and E. cichoracearum DC on cucumber leaves. Urquhart and Punja (1997, 2002) investigated the effect of T. pallescens on Sphaerotheca fuliginea on cucumber leaves. Ng and MacDonald (1997) report on the biological control of rose powdery mildew by T. pallescens. Haggag et al. (2012) controlled powdery mildew of mango by T. pallescens in pilot trials. Activity of β-1,3-glucanase produced by Tilletiopsis spp. was first detected by Urquhart et al. (1994). Urquhart and Punja (2002) studied exo- and endo-β-1,3-glucanase and chitinase production and considered that hydrolytic enzymes in combination with fatty acids are involved in the antagonism of Tilletiopsis spp. against powdery mildew pathogens. Haggag et al. (2015) investigated the mode of action of an isolate of T. pallescens antagonistic to powdery mildew in grapevine and reported on the secretion of proteases by the antagonist. Tilletiospis spp. have not been reported as plant pathogens. However, white haze, a superficial skin discoloration, on apple caused by Tilletiopsis spp., has been reported recently for some regions. In conclusion, a set of antagonists against Bgt was found in a screening program in less than three years following a stepwise screening approach. More than 1200 isolates from powdery mildew pustules were assessed in an initial screening focussing on ecological characteristics, sporulation capacity and safety issues. Selected isolates were subsequently tested in bioassays and the efficacy of the most promising isolates was confirmed under field conditions. Research on production and formulation of blastospores of the selected T. pallescens isolates has been initiated to test their efficacy in powdery mildew control at field scale in wheat. Other crops infected by different powdery mildew pathogens will be included in these studies. A stepwise screening approach is a powerful strategy to find new

microbial biological control agents against diseases and pests with an efficient use of resources and a considerable chance to develop commercially viable solutions. Depending on the targeted diseases and pests, relevant sources of potential antagonists have to be identified and specific selection criteria have to be used for certain selection steps. For example, for seed treatments major additional criteria are resistance of antagonists against drying processes after application to seeds, shelf life on surfaces of treated seeds and rapid colonization of seedling rhizospheres after sowing. CRediT authorship contribution statement Jürgen Köhl: Conceptualization, Methodology, Validation, Formal analysis, Visualization, Writing - original draft, Writing - review & editing, Funding acquisition. Helen Goossen-van de Geijn: Methodology, Investigation. Lia Groenenboom-de Haas: Methodology, Investigation, Writing - review & editing. Betty Henken: Methodology, Investigation. Rüdiger Hauschild: Conceptualization, Methodology, Investigation, Writing - original draft, Funding acquisition. Ulrike Hilscher: Methodology, Investigation. Carin Lombaersvan der Plas: Methodology, Investigation, Writing - review & editing. Trudy van den Bosch: Methodology, Investigation. Mariann Wikström: Methodology, Investigation, Writing - review & editing, Funding acquisition. Acknowledgements The project BIOCOMES has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 612713 and the Dutch Ministry of Agriculture, Nature and Food Quality (Programme Durable Plant Production, project BO-25.10-001-002). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biocontrol.2019.104008. References Alabouvette, C., Olivain, C., Migheli, Q., Steinberg, C., 2009. Microbial control of soilborne phytopathogenic fungi with special emphasis on wilt-inducing Fusarium oxysporum. New Phytol. 184, 529–544. https://doi.org/10.1111/j.1469-8137.2009. 03014.x. Anonymous, 2009. Regulation (EC) No. 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. Off. J. Eur. Union L 309, 1–50. Askary, H., Carriere, Y., Bélanger, R.R., Brodeur, J., 1998. Pathogenicity of the fungus Verticillium lecanii to aphids and powdery mildew. Biocontrol Sci. Techn. 8, 23–32. https://doi.org/10.1080/09583159830405. Bensch, K., Groenewald, J.Z., Dijksterhuis, J., Starink-Willemse, M., Andersen, B., Summerell, B.A., et al., 2010. Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales). Stud. Mycol. 67, 1–94. https://doi.org/10.3114/sim.2010.67.01. Bensch, K., Braun, U., Groenewald, J.Z., Crous, P.W., 2012. The genus Cladosporium. Stud. Mycol. 72, 1–401. https://doi.org/10.3114/sim0003. Boekhout, T., 2011. Tilletiopsis Derx ex Derx. In: Kurtzman, C., Fell, J.W., Boekhout, T. (Eds.), The Yeasts, 5th edn. Elsevier Science, Amsterdam, pp. 2003–2014. Campbell, G.S., Gardner, W.H., 1971. Psychrometric measurement of soil water potential: temperature and bulk density effects. Soil Sci.. Soc. Am. J. 35, 8–12. https://doi.org/ 10.2136/sssaj1971.03615995003500010011x. Cao, X., Duan, X., Zhou, Y., Luo, Y., 2012. Dynamics in concentrations of Blumeria graminis f. sp tritici conidia and its relationship to local weather conditions and disease index in wheat. Eur. J. Plant Pathol. 132, 525–535. https://doi.org/10.1007/s10658011-9898-8. Carbone, I., Kohn, L.M., 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91, 553–556. https://doi.org/10.2307/ 3761358. Dik, A.J., Verhaar, M.A., Bélanger, R.R., 1998. Comparison of three biological control agents against cucumber powdery mildew (Sphaerotheca fuliginea) in semi-commercial-scale glasshouse trials. Eur. J. Plant Pathol. 104, 413-/423. https://doi.org/10. 1023/A: 1008025416672.

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