Suppression of carpogenic germination and viability of Sclerotinia sclerotiorum sclerotia by biofumigation, biological control and Perlka

Suppression of carpogenic germination and viability of Sclerotinia sclerotiorum sclerotia by biofumigation, biological control and Perlka | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Suppression of carpogenic germination and viability of Sclerotinia sclerotiorum sclerotia by biofumigation, biological control and Perlka Madhavi P. Dassanayaka, Seona G. Casonato, Eirian Jones This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5339317/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Sclerotia play an important role in the disease cycle of Sclerotinia sclerotiorum . Traditional control measures do not effectively suppress germination and viability of sclerotia. This study performed a series of in-vivo bioassays to investigate the effect of biofumigants produced by Brassica juncea ‘Caliente 199’, biological control by Coniothyrium minitans, and Perlka â on carpogenic germination and sclerotial viability. Coniothyrium minitans alone or in combination with Perlka â resulted in complete inhibition of carpogenic germination and high sclerotial mortality. Perlka â treatment in quantities equivalent to field rates of 400 and 300 kg ha -1 resulted in 100% and 77% reduction in sclerotial germination, respectively compared with the untreated control, but viability remained high (³97.5%). Treating the potting mix with plant tissue of ‘Caliente 199’ in quantities equivalent to the field rate of 100 and 75 t ha -1 reduced carpogenic germination by about 46% compared to unamended control but did not result in significant reduction in sclerotial viability. Coniothyrium minitans and Perlka â were effective in suppressing carpogenic germination but the suppression achieved with biofumigation was much lower. Coniothyrium minitans with Perlka â resulted in high sclerotial mortality, which was not achieved with the Perlka â only treatment. These results showed that of the treatments tested, C. minitans able to reduce the carpogenic germination and the viable inoculum. Further studies are needed to assess the effectiveness and potential of integrating with non-pesticide methods to achieve better control of S. sclerotiorum in the field. Brassica juncea ‘Caliente 199’ Coniothyrium minitans integrated pest management (IPM) calcium cyanamide. Figures Figure 1 Introduction Sclerotinia sclerotiorum is a ubiquitousplant pathogenic fungus causing substantial yield losses in a broad range of hosts around the world (Boland & Hall, 1994; Bolton et al., 2006; Purdy, 1979). The pathogen is recorded to infect more than 400 plant species from 278 genera belonging to 75 families (Boland & Hall, 1994), with infection of susceptible hosts resulting in up to 100% yield loss (Purdy, 1979; Saharan & Mehta, 2008). Sclerotinia sclerotiorum is a devastating pathogen in a range of plants in New Zealand (Rabeendran, 2000) including lettuce, canola, beans, peas, potatoes, peanuts, carrots, sweet potatoes, kiwifruit and many other vegetable crops (Broadhurst et al., 1997; Hoyte et al., 2007; Lewthwaite & Wright, 2005; Pennycook, 1985, 1989). Sclerotia play a crucial role in the disease cycle of S. sclerotiorum as they act as inocula and survival structures (Bolton et al., 2006; Smolińska & Kowalska, 2018; Willetts & Wong, 1980). Sclerotia can germinate myceliogenically to infect the lower plant parts directly (Bardin & Huang, 2001; Bolton et al., 2006). Alternatively, apothecia produced by carpogenic germination of sclerotia subsequently produce ascospores, which cause infection of aboveground and soil-line plant tissues (Abawi & Grogan, 1979; Bolton et al., 2006). The sclerotia can remain viable in soil for at least 5 years (Adams & Ayers, 1979; Peltier et al., 2012; Wu & Subbarao, 2008) which makes management of the pathogen in the field more challenging (Smolińska & Kowalska, 2018). Conventional control measures such as using fungicides are largely ineffective at reducing carpogenic germination and/or sclerotial viability. Therefore, research to investigate sustainable control options have been pursued. Moreover, environmentally friendly methods are increasingly being sought due to the potential adverse impacts on the environment and the development of resistance in the pathogen population due to the continuous application of chemical control measures. Biofumigation is the process of incorporating macerated plant material of Brassicaceae crops into the soil which, through the hydrolysis of glucosinolate compounds (GSLs) in their plant tissues, results in the production and release of isothiocyanates (ITCs) which can suppress soilborne pests and pathogens (Angus et al., 1994; Kirkegaard et al., 1993; Kirkegaard & Sarwar, 1998). The term biofumigation has also been expanded to include other plant species such as Allium spp. and Sorghum spp. which also produce biotoxic compounds with comparable suppression to that achieved by Brassicaceae plants (Reddy, 2013). Isothiocyanate compounds released in the biofumigation process of Brassica spp. have been reported to suppress many soilborne fungi including S. sclerotiorum (Larkin & Lynch, 2018; Ojaghian et al., 2012; Smolinska & Horbowicz, 1999; Warmington & Clarkson, 2016). Research reports also indicate the potential of synthetic ITCs or ITCs liberated from Brassica tissues to suppress the carpogenic germination and/or viability of S. sclerotiorum sclerotia (Dandurand et al., 2000; Dassanayaka et al., 2023; Kurt et al., 2011; Warmington & Clarkson, 2016). Among the many identified mycelial and sclerotial antagonists of S. sclerotiorum , Coniothyrium minitans (reclassified as Paraconiothyrium minitans by Verkley et al. (2004) but referred to as C. minitans in this paper) is a widely studied sclerotial parasite and is commercially available as a biological fungicide. Coniothyrium minitans hasbeen utilised as a biocontrol agent in many bioassays and field-based studies and has been demonstrated to suppress the carpogenic germination and viability of S. sclerotiorum sclerotia (Gerlagh et al., 2003; Jones et al., 2003; McQuilken et al., 1995; Zeng et al., 2012). However, its incorporation with other control strategies has not been widely investigated. Perlka® (calcium cyanamide) is a multi-functional fertilizer which is used as a soil amendment. Calcium cyanamide in Perlka â is known to have a direct fungicidal effect as a soil disinfectant, similar to methyl bromide (Bletsos, 2006; Bourbos et al., 1997; Wang et al., 2007) and indirect effects on controlling soilborne pathogens (Huang & Sun, 1991; Liu et al., 2016; Tremblay et al., 2005). Cyanamide in Perlka â has been shown to be effective in suppressing the carpogenic germination of sclerotia of S. sclerotiorum (Huang et al., 2006; Huang & Sun, 1991; Warmington & Clarkson, 2016) but its effect on the viability of sclerotia is unclear. Administering more than one control measure with different modes of action to target the pathogen in an integrated pest management (IPM) approach rather than relying on a single control measure is recommended. To enable an IPM strategy to be designed to provide effective control of S. sclerotiorum further information is required on the effectiveness of the aforementioned alternative non-pesticide control measures to suppress inoculum production by this pathogen. Therefore, the current study investigated; (1) the potential of biofumigants produced by Brassica juncea ‘Caliente 199’ (referred to as ‘Caliente 199’ henceforth), biological control by C. minitans, and Perlka â to reduce carpogenic germination and sclerotial viability, and (2) the effect of C. minitans and Perlka â as a combined application on the carpogenic germination and sclerotial viability of S. sclerotiorum . Materials and Methods Preparation of fungal inoculum Sclerotinia sclerotiorum isolates LUPP479 and LUPP2650 (NCBI accession number OROR876349 and OR855935, respectively) originating from New Zealand were obtained from the Lincoln University Plant Pathology (LUPP) culture collection and selected for the experiments. Isolate LUPP479 was recovered from cauliflower ( Brassica oleracea ) in 1996 and originated in Canterbury (South Island, New Zealand) and isolate LUPP2650 which originated in Auckland (North Island, New Zealand) was recovered from lettuce ( Lactuca sativa ) in 2017. Based on the carpogenic germination results of the previous studies (Dassanayaka, 2023), sclerotia of LUPP475 showed greater germination by subjecting to 4°C preconditioning treatment (conditioned sclerotia) whilst sclerotia of LUPP2650 germinated irrespective of preconditioning. Hence, conditioned sclerotia of LUPP479 and LUPP2650 (in order to maintain the same pretreatment conditions) were used for the initial experiment with biofumigants and unconditioned sclerotia of LUPP2650 was used for other experiments. The sclerotia were produced on sterilized wheat grain following the procedure described by Jones et al. (2003). Conical flasks (250 mL) containing 25 g of wheat grain moistened with 50 mL distilled water were sterilized by autoclaving (121°C, 30 min) twice on consecutive days. Sterilized wheat was inoculated with three S. sclerotiorum colonised PDA (Potato Dextrose Agar, Difco TM ) discs (Ø 7 mm) cut from the margins of an actively growing 3-day-old fungal culture. Inoculated flasks were incubated for 4 weeks at 20°C in 24 h dark. Conditioned and unconditioned sclerotia were produced by extended incubation of sclerotia at 4°C and 20°C respectively for another 4 weeks in 24 h dark. Sclerotia were harvested by washing the contents of the flask (wheat and sclerotia) with sterile distilled water. Sclerotia of 2-4 mm in diameter were collected by sieving the contents through a 4 mm sieve and collecting them onto a 2 mm sieve. The sclerotia were placed on sterile paper towels and air-dried inside a laminar flow hood overnight. The sclerotia were further sorted manually to select the sclerotia of uniform size, shape and an intact rind before using them for the experiment. A new batch of sclerotia was produced for each experiment and 10 randomly selected sclerotia were cultured on PDA to ascertain their viability based on production of mycelium. In-vivo bioassays for suppression of carpogenic germination of sclerotia Effect of ‘Caliente 199’ biofumigants An in-vivo bioassay was set up to test the effect of biofumigants released by macerated plant tissue of ‘Caliente 199’ on carpogenic germination of conditioned sclerotia of two S. sclerotiorum isolates, LUPP479 and LUPP2650 in sterile potting mix. The soil containing potting mix was prepared based on the modification of John Innes compost No. 1 (https://www.gardeningdata.co.uk/soil/john_innes/john_innes.php). The composition of the potting mix contains 0.58 L of loam, 0.25 L of peat, 0.17 L of sand, mixed with 0.6 g of ground limestone, 1.2 g of Osmocote® (38-0-0), 1.2 g of superphosphate and 0.6 g of potassium sulphate (pH 5.8). The fresh batch of potting mix was prepared and sterilized by autoclaving (121°C, 30 min) before setting up the experiment. Whole plant tissues of ‘Caliente 199’ at 50% flowering was tested at three rates equivalent to field biomass rates of 50, 75 and 100 t ha -1 . The weights of fresh biomass used to achieve these field rates (considering an incorporation depth of 15 cm) were calculated as 3.35 g, 5.02 g and 6.70 g, respectively to treat 100 mL of potting mix. Clear plastic cylindrical containers (250 mL) were filled with 100 mL of potting mix (approximately 94 g) and the required quantity of macerated plant tissue and mixed thoroughly to the total volume of the potting mix in the container. Controls consisted of unamended potting mix. To avoid any loss of volatile biofumigants from the plant material, all the replicates of one treatment were set up together before moving to the next treatment. For each container, five sclerotia were gently pressed into the surface of the potting mix in a grid pattern with the weight of the five sclerotia measured and recorded before placement in the substrate. The moisture of the potting mix was adjusted to 30% (w/w) by adding sterile distilled water and the weight of the container was recorded (without the lid). The containers were sealed by replacing the lid immediately after adding water. After 2 weeks, the screw caps of the containers were replaced with clear polythene wrappers (40 mm thickness, Bio-strategy Ltd) for better light penetration. The moisture level was maintained by watering with sterile distilled water every two weeks to bring the weight of the containers back to their original weight. The containers were incubated at 18°C under 14L:10D light regime (Warmington & Clarkson, 2016) within an incubator (Conviron Ò model CMP6010). Eight replicates of each treatment and controls were set up in a randomized complete block design (RCBD). The development of stipes and/or apothecia was monitored and recorded every week for up to 15 weeks. After 15 weeks, the ungerminated sclerotia were recovered and surface sterilized with 1:1 (v:v) solution of ethanol: bleach (with 5.3% w/v NaOCl) for 3 min followed by washing with sterile distilled water three times (Jones et al., 2003). Sclerotia were bisected and plated on PDA and assessed for sclerotial viability based on the mycelial germination of sclerotia. Effect of Perlka â A box bioassay was set up to test the effect of Perlka â (Alzchem) at two different field rates (300 and 400 kg ha -1 ) on the carpogenic germination of unconditioned sclerotia of S. sclerotiorum isolate LUPP2650. Clear plastic boxes (120 x 120 x 90 mm) were filled with approximately 500 mL (~450 g) of the sterile soil-based potting mix prepared as previously described. The equivalent quantities to achieve the two field rates of Perlka â , 300 and 400 kg ha -1 , were calculated considering the depth of incorporation (10 cm) for 500 mL of soil at 0.20 g and 0.15 g for each box. Initially, the boxes were partially filled (up to 4 cm) and Terylene mesh bags (<2 mm pore size and 5 x 5 cm in size) containing 5 sclerotia inside each bag and heat sealed, were placed on the surface of the potting mix. The measured quantities of Perlka â were added and mixed with the remaining potting mix (approximately 150 g) and placed on top of the mesh bags containing the sclerotia to fill the boxes. This resulted in the sclerotial bags being buried approximately 2 cm from the top surface level of the potting mix. The potting mix containing no Perlka â was used as the control. Five free individual sclerotia were placed and pressed into the top of the substrate surface. The moisture of the potting mix was adjusted to 30% w/w by adding sterile distilled water, the lid was replaced, and the weight of the box was recorded. The moisture was maintained by watering with sterile distilled water every two weeks to maintain the weight of each box to its original weight. The boxes were incubated at 18ᵒC under a 14L:10D light regime inside an incubator. Eight replicates of each treatment and control were set up in a RCBD. Stipes and apothecial development were monitored every week for up to 15 weeks. After 15 weeks, the sclerotia in the mesh bags were recovered, and tested for their viability as described previously. Effect of C. minitans and Perlka â A box bioassay was done to test the effect of Perlka â and C. minitans when applied alone and in combination, on the carpogenic germination of unconditioned sclerotia of S. sclerotiorum isolate LUPP2650. Clear plastic boxes filled with approximately 500 mL of soil based potting mix (~450 g) were used for the experiment. Based on the initial screening of 10 different C. minitans isolates originating from New Zealand for their in vitro sclerotial parasitism, isolate LUPP418 (isolated from sclerotia of Sclerotinia trifoliorum ) which showed the highest sclerotial parasitism (Fig. 1a) was used in this experiment (Table SI1 and Fig. SI1). The conidial suspension of C. minitans (LUPP418) was prepared by adapting the method described by Jones et al. (2014). A 21-day-old culture was flooded with 10 mL of sterile distilled water and the surface mycelium was scraped using a sterile spreader to dislodge the conidia. The resulting conidial suspension was strained through a double layer of sterile Miracloth to remove any mycelial fragments. The spore concentration was determined using a haemocytometer and 750 mL of 10 7 spores mL -1 spore suspension was prepared. In parallel, the spore suspension was diluted to make 10 3 conidia mL -1 and 100 mL of the suspension was plated on PDA to test for spore viability and germinability. The prepared spore suspension was added to 7.5 kg of the sterile potting mix and mixed thoroughly throughout the entire volume of the potting mix in a 30 L clean plastic box resulting in a final spore concentration of 10 6 g -1 potting mix. The C. minitans amended potting mix was used to fill 16 boxes, with 450 g added to each. Of the 16 boxes, 8 were treated additionally with Perlka â by mixing the measured Perlka â equivalent to the field rate of 400 kg ha -1 to the top 2 cm layer of the potting mix, as explained previously. A further 7.5 kg of the potting mix was treated with 750 mL of sterile distilled water instead of the spore suspension, as above, and 16 boxes were filled with 450 g each. Of the 16 boxes, 8 were additionally treated with Perlka â of the field rate of 400 kg ha -1 , and the remaining untreated 8 boxes were considered the control. In each box, Terylene mesh bags (<2 mm pore size and 5 x 5 cm in size) containing 5 sclerotia were buried within the top 2 cm of potting mix and five free individual sclerotia were pressed into the surface of the potting mix as described previously. The boxes were incubated at 18ᵒC under a 14L:10D light regime inside an incubator. Eight replicates of each treatment and control were set up in a RCBD. Stipes and apothecial development were monitored every week for up to 15 weeks. After 15 weeks, the sclerotia in the mesh bags were recovered, and tested for their viability on PDA following the similar procedure explained in the previous experiment. Statistical analysis The number of germinated sclerotia (sclerotia which produced at least one stipe), the number of apothecia per sclerotium and the time to produce the first apothecium by individual sclerotia, were recorded in all three experiments. The percentage data (% sclerotial germination) were arcsine square root transformed before the analyses. Data were analysed using general analysis of variance (ANOVA) with mean separated using post-hoc Tukey’s HSD tests at P = 0.05. The total weight of the five free sclerotia placed in each replicate was used as a covariate in the biofumigant experiment. Values are presented as back transformed means. All the analyses were done using GenStat22 software (VSN international, Hemel Hempstead, United Kingdom). Results Effect of ‘Caliente 199’ biofumigants The sclerotial germination of both isolates (LUPP479 and LUPP2650) was observed in the potting mix amended with ‘Caliente 199’ (Fig. 1b) and unamended controls. The effect of the quantity of plant material was significant ( P <0.001) but not the isolate ( P =0.973) on the percentage carpogenic germination of sclerotia. The potting mix treated with ‘Caliente 199’ in the quantities equivalent to the field rates of 75 and 100 t ha -1 resulted in ~46% less carpogenic germination of sclerotia and this was significantly less ( P < 0.001) than the unamended control (Table 1). Treating the potting mix with ‘Caliente 199’ at a quantity equivalent to the field rate of 50 t ha -1 resulted in similar carpogenic germination to the unamended control and the germination was greater by 41% compared with the sclerotia in potting mix treated with quantity equivalent to 100 t ha -1 . For the sclerotia which had germinated, the mean number of apothecia per sclerotium varied from 1.6 to 2.5 across both isolates irrespective of the quantity of the biofumigant material. The effect of biofumigant released from different field rates of ‘Caliente 199’ plant tissue significantly ( P < 0.001) affected the days to development of the first apothecium by sclerotia. The sclerotia in potting mix amended with the quantity equivalent to the field rates of 75 t ha -1 and 50 t ha -1 , produced the first apothecium, 9 - 11 days earlier, compared with the sclerotia in the untreated control. The viability of the non-germinated sclerotia of both isolates recovered from all the biofumigant treatments and unamended controls at the end of the experiment was high, being ³91.0% for both. Effect of Perlka â Although most of the sclerotia in the Perlka â treated potting mix produced stipe initials, none in the potting mix treated with 400 t ha -1 and only a few sclerotia in the potting mix treated with 300 t ha -1 Perlka â developed fully elongated stipes or apothecia. Only sclerotia which developed stipes or apothecia were considered to have carpogenically germinated. Amending the potting mix with Perlka â equivalent to 400 and 300 t ha -1 resulted in significantly ( P < 0.001) lower carpogenic germination, with 400 t ha -1 Perlka â completely inhibiting germination and 300 t ha -1 Perlka â resulting in 79.6% reduction compared with the unamended control which showed 83.0% carpogenic germination (Table 2) . The numbers of apothecia per sclerotium produced in the unamended potting mix (Fig. 1c) and potting mix amended with Perlka â at 300 t ha -1 were 2.4 and 2.1, respectively and did not differ significantly ( P = 0.659) from each other. The sclerotia in the amended potting mix amended with Perlka â at 300 t ha -1 took 10 more days to develop the first apothecium than the unamended control, however they did not differ significantly ( P = 0.109) from each other. Stipe initials, which is the initial stage of apothecial formation, were observed in the sclerotia recovered from the mesh bags which were buried in the potting mix treated with Perlka â and controls (unamended) indicating sclerotia were viable. Further, sclerotia produced fungal mycelium characteristics of S. sclerotiorum on PDA confirming their viability (Fig. 1d) . Perlka â did not reduce the viability of sclerotia, where the viability of buried sclerotia in the treatments and control was ³97.5%. Effect of C. minitans and Perlka â Complete inhibition of carpogenic germination of sclerotia was observed for either C. minitans or Perlka â applied alone, or a combined application of C. minitans and Perlka â , whilst the sclerotial germination in the unamended control was 83.0 ± 4.5% (Table 3). When recovering the buried sclerotia from the potting mix treated with C. minitans , it was observed that the sclerotia were disintegrating and clumped together (Fig. 1e) . The inside tissue (medulla) of most of the sclerotia were degraded leaving only the outer rind. Additionally, at the time of surface sterilising and subsequent culturing on PDA, the sclerotia were noted to be friable. After transferring to PDA, most of the sclerotia failed to develop S. sclerotiorum mycelium. Instead, other bacterial and fungal contaminations were observed growing from the sclerotia indicating non-viability. The development of mycelium and pycnidia of C. minitans on a few sclerotia was observed (Fig. 1f) . The sclerotia buried in the potting mix treated with C. minitans only and combined with Perlka â , had reduced sclerotial viability, with 5.0% and 2.5%, respectively. A greater percentage of sclerotia (³95%) in the potting mix treated with only Perlka â , and the untreated control showed mycelial growth of S. sclerotiorum on PDA indicating they are viable but no infection by C. minitans was observed for any of the sclerotia. Discussion Of the different treatments assessed to suppress the carpogenic germination of S. sclerotiorum sclerotia in the in vivo bioassays of this study, the antagonism by C. minitans resulted in the greatest suppression of both carpogenic germination and sclerotial viability. Perlka â was successful in reducing the carpogenic germination but sclerotial viability mostly remained unaffected. Biofumigation released from ‘Caliente 199’ plant tissue in contrast resulted in less suppression of carpogenic germination with sclerotia mostly remaining viable. The higher quantities of ‘Caliente 199’ at the field rates (100 and 75 t ha -1 ) inhibited the carpogenic germination of S. sclerotiorum sclerotia by approximately 46% compared with the unamended control. However, the quantity equivalent to the field rate of 50 t ha -1 did not result in a significant reduction in carpogenic germination. In agreement with these results, Warmington and Clarkson (2016) showed that the dried milled plant material of B. juncea ‘Caliente 99’ incorporated into compost at the field rate of 100 t ha -1 resulted in ~45% reduced carpogenic germination compared with the untreated control after 150 days. Brassica juncea has the GSL profile of 2-propenyl (sinigrin), 2- phenylethyl and benzyl (Kirkegaard et al., 1998). These GSLs produce aliphatic allyl ITC, and aromatic 2- phenylethyl and benzyl ITCs upon the enzymatic breakdown. Brassica juncea ‘Caliente 199’ used in the present study is a commercial biofumigant mustard blend which has been reported to produce high levels of sinigrin (Doheny-Adams et al., 2017; Duff & Firrell, 2021). Hence, allyl ITC, along with other ITCs, are likely to have inhibitory activity on the carpogenic germination of the sclerotia. The setup of the bioassay was done as a contact phase experiment in which both aliphatic and aromatic ITCs may be active on the sclerotia and reduce carpogenic germination. It was also clear that increasing the quantity of plant material resulted in an increased inhibition, most likely linked to the greater concentration of ITCs released and available to act on the sclerotia in the potting mix but were not enough to achieve a complete inhibition. Dandurand et al. (2000) however demonstrated complete inhibition of carpogenic germination of S. sclerotiorum sclerotia in sterile soil amended with defatted seed meal of B. napus cv. Dwarf Essex. The comparatively higher inhibition of carpogenic germination reported in the study of Dandurand et al. (2000) than in the present study is most likely due to B. napus having a different GSL profile which may be more effective in suppressing the sclerotial germination. In addition, the use of seed meal may also have increased the efficacy of inhibition, where defatting concentrates the GSLs in seeds (Serrano-Pérez et al., 2021). Further, the incorporation of ‘Caliente 199’ tissue of lower field rates resulted in the early development of apothecia compared with the unamended control. This is possibly due to the toxic bioactive compounds weakening the sclerotial rind and leading to the acceleration of apothecial production. Also, early apothecial production may be a stress response against the toxic bioactive compounds produced by ‘Caliente 199’ tissue, which promoted the fungus to reproduce and sporulate. There are no comparable previous studies with S. sclerotiorum , but induced sporulation in the presence of high concentration of a fungicide (difenoconazole) has been demonstrated in Colletotrichum spp. by Han et al. (2022). The viability of sclerotia in the biofumigant treatments was ³90% and indicates that the bioactive compounds produced by ‘Caliente 199’ tissue are not effective enough to kill the sclerotia. This further confirms the results of Dassanayaka et al. (2023) where exposure of sclerotia to ‘Caliente 199’ plant tissue in a volatile phase experiment did not result in significant reduction in sclerotial viability. Amending the potting mix with Perlka â resulted in 80 - 100% suppression of carpogenic germination but no substantial reduction in the sclerotial viability. These results also concur with those of Warmington and Clarkson (2016) whereby the mean carpogenic germination of S. sclerotiorum sclerotia was reduced by 93% when incubated in pasteurized potting mix treated with 400 kg ha -1 Perlka â , compared with the untreated control. The partial decomposition of calcium cyanamide in Perlka â results in the release of cyanamide (Huang & Sun, 1991) urea and ammonia (Huang & Janzen, 1991), which are reported to have an inhibitory effect on the carpogenic germination of S. sclerotiorum sclerotia. In the current study, although most of the sclerotia formed stipe initials they did not further differentiate into stipes or apothecia. Hence these compounds seem to exert an inhibitory effect on apothecial development rather than directly killing sclerotia. The application rates used in the current study were clearly not effective to kill the sclerotia. Huang and Sun (1991) reported complete inhibition of carpogenic germination of sclerotia of S. sclerotiorum in autoclaved soil treated with Perlka â at the rates of 0.6, 1% and 2% (w/w) and 100% reduction in viability of buried sclerotia at all three application rates. The application rates of Perlka â in the study of Huang and Sun (1991) were much higher compared to the present study (0.07% and 0.09% w/w) and are likely to account for the greater reduction in the sclerotial viability. Wright (2011) however demonstrated that the viability of S. minor sclerotia remained high (73% and 94% viable sclerotia) after application of Perlka â at the rate of 500 kg ha -1 in two small-scale lettuce field studies. Hence, it is important to understand the efficacy of Perlka â to suppress the pathogen at the recommended field rates (300 -500 kg ha -1 , (Alzchem, 2023) under field conditions and warrants further studies. Complete inhibition of carpogenic germination of sclerotia was achieved by treating the potting mix with the conidial suspension of C. minitans alone, with almost complete reduction in sclerotia viability. Coniothyrium minitans invades sclerotia via enzymatic hydrolysis and mechanical pressure (Huang & Kokko, 1988). The hypha of the antagonist then grows into the sclerotia and degrades the cells in the cortex and medulla though the production of extracellular enzymes like chitinase and b-1,3 glucanase (Jones & Watson, 1969). It was observed in this study that the buried sclerotia parasitized with C. minitans became soft and disintegrated leaving only the outer rind. The sclerotia parasitised by C. minitans failed to produce apothecia and subsequently disintegrated. The results of the present study agree with the results of the pot bioassay done by Jones et al. (2014) where no apothecia were produced by sclerotia inoculated with a spore suspension of C. minitans (10 6 spores mL -1 ) and placed in pots containing cabbage seedlings over the 20 week experimental period. The results also concur with the study done by Bennett et al. (2005) that showed greater S. sclerotiorum sclerotial mortality of 90.7% in non-sterilized soil treated with C. minitans (10 6 cfu g -1 of soil) compared with the untreated control (0% sclerotial mortality) after 90 days incubation. Not only does the sclerotial parasitic activity of different C. minitans isolates vary,but the efficacy of a C. minitans isolate to parasitize different S. sclerotiorum isolates has also been shown to vary (Jones & Stewart, 2000). Further, the efficacy of sclerotial parasitism also depends on the incubating conditions or soil conditions in the field (de Vrije et al., 2001; Jones et al., 2004). Hence, understanding the potential efficacy of the C. minitans isolate tested in this study under field conditions is essential to determine the potential for developing a commercial formulation for incorporation in an IPM programme to control the pathogen. Applying C. minitans in combination with Perlka â resulted in complete inhibition of carpogenic germination and high sclerotial mortality similar to the level achieved with the application of C. minitans alone. Hence, it can be concluded that the sclerotial mortality is predominantly due to the antagonistic activity of C. minitans but importantly there was no negative effect from Perlka â on the sclerotial parasitic activity by C. minitans . Since both Perlka â and C. minitans alone resulted in complete inhibition of carpogenic germination, any synergistic effect could not be assessed. But Yang et al. (2011) demonstrated a synergistic effect from the combined application of C. minitans with a compound fertilizer containing N: P 2 O 5 :K 2 O at 15:15:15 in a pot experiment. Whether lower inoculum concentrations of C. minitans and Perlka â would result in a synergistic effect should be tested in the field, as it might provide a more economic approach to the effective control of S. sclerotiorum disease in the field. The results of the study further confirmed the previous literature showing the potential to control S. sclerotiorum by the non-pesticide methods tested. Successful suppression of carpogenic germination or control of Sclerotinia disease by C. minitans applicationunder field or similar conditions has been reported by several studies (Chitrampalam et al., 2010; Huang & Erickson, 2000; Jones et al., 2014). The field application of Perlka â has also shown promising results in suppressing apothecial production (Huang et al., 2006). Although the biofumigation treatment with ‘Caliente 199’ did not result in appreciable suppression of sclerotial germination, our previous study reported ‘Caliente 199’ to inhibit the mycelial growth of S. sclerotiorum (Dassanayaka et al., 2023) and therefore may have activity to prevent mycelial infection of plants by S. sclerotiorum . Additionally, there are other biofumigant crop types which have been reported to suppress carpogenic germination of S. sclerotiorum including Raphanus sativus (Warmington & Clarkson, 2016), B. campestris (Ojaghian et al., 2012) and B. napus (Dandurand et al., 2000) that could be tested in an integrated approach. Coniothyrium minitans and Perlka â were equally effective in suppressing carpogenic germination under the tested conditions. Incorporating C. minitans along with Perlka â resulted in substantial sclerotial mortality which was not achieved by the Perlka â alone per se. This indicates the potential of C. minitans to reduce the amount of viable inoculum in the soil and its importance in an IPM program to control the pathogen. The synergistic effect could not be assessed however with the concentration used and future studies could assess with lower concentrations of both components for their potential to provide enhanced suppression of sclerotial germination and viability. Also, there is a potential to integrate these non-pesticide applications with biofumigation or even with reduced application of fungicide. The efficacy of any control measure in the field is influenced by many environmental factors and soil conditions. Therefore, further studies are warranted to assess these control measures alone or as integrated applications to control Sclerotinia disease of crop plants under glasshouse and field conditions. The results of the study can be used in the development of an IPM package to achieve sustainable control of S. sclerotiorum which is an intractable plant pathogenic fungus. Statement and Declarations Acknowledgements First author acknowledges a PhD scholarship awarded by the Accelerating Higher Education and Development operation (AHEAD), Sri Lanka. The authors also gratefully acknowledge an internal grant from the Agriculture and Life Sciences Faculty, Lincoln University to support the research. The authors are grateful to Dr Peter Wright (The New Zealand Institute for Plant and Food Research Limited) for contributing isolates. The authors acknowledge Sandy Hammond for her assistance in the laboratory experiments. Data availability Data available on request. Funding The work was funded by an internal grant from Lincoln University. Competing Interest: No competing interest to declare. Ethics approval: All the ethical standards have been followed and no human or animal testing was involved in the experiments. Consent to participate: Not applicable. Consent for publication: Consent for publication was obtained from all co-authors. Conflicts of interest: There are no conflicts of interest to declare. Author Contributions: Madhavi P. Dassanayaka : Conceptualisation, Methodology, Investigation, Formal Analysis, Writing – Original Draft, Writing - Review and Editing and Visualization. Seona G. Casonato : Conceptualisation, Methodology, Formal Analysis, Supervision, Writing - Review and Editing, Validation and Visualization. E. Eirian Jones : Conceptualisation, Methodology, Supervision, Project Administration and Funding Acquisition, Supplying the Resources, Writing - Review and Editing, Validation and Visualisation. References Abawi, G. S., & Grogan, R. G. (1979). Epidemiology of diseases caused by Sclerotinia species. Phytopathology , 69 , 899-904. https://doi.org/10.1094/Phyto-69-899 Adams, P. B., & Ayers, W. A. (1979). Ecology of Sclerotinia species. Phytopathology , 69 , 896-899. https://doi.org/10.1094/Phyto-69-896 Alzchem. (2023). Application rate of Perlka® . Retrieved 7th October from https://www.alzchem.com/en/brands/perlka/application-area/ Angus, J. F., Gardner, P. A., Kirkegaard, J. A., & Desmarchelier, J. M. (1994). Biofumigation: isothiocyanates released from brassica roots inhibit growth of the take-all fungus. Plant and Soil , 162 (1), 107-112. https://doi.org/10.1007/BF01416095 Bardin, S. D., & Huang, H. C. (2001). Research on biology and control of Sclerotinia diseases in Canada. Canadian Journal of Plant Pathology , 23 (1), 88-98. https://doi.org/10.1080/07060660109506914 Bennett, A. J., Leifert, C., & Whipps, J. M. (2005). Effect of combined treatment of pasteurisation and Coniothyrium minitans on sclerotia of Sclerotinia sclerotiorum in soil. European Journal of Plant Pathology , 113 (2), 197-209. https://doi.org/10.1007/s10658-005-2331-4 Bletsos, F. A. (2006). Grafting and calcium cyanamide as alternatives to methyl bromide for greenhouse eggplant production. Scientia Horticulturae , 107 (4), 325-331. https://doi.org/10.1016/j.scienta.2005.09.003 Boland, G. J., & Hall, R. (1994). Index of plant hosts of Sclerotinia sclerotiorum . Canadian Journal of Plant Pathology , 16 (2), 93-108. https://doi.org/10.1080/07060669409500766 Bolton, M. D., Thomma, B. P., & Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Molecular Plant Pathology 7 (1), 1-16. https://doi.org/10.1111/j.1364-3703.2005.00316.x Bourbos, V. A., Skoudridakis, M. T., Darakis, G. A., & Koulizakis, M. (1997). Calcium cyanamide and soil solarization for the control of Fusarium solani f.sp. cucurbitae in greenhouse cucumber. Crop Protection , 16 (4), 383-386. https://doi.org/10.1016/S0261-2194(96)00110-X Broadhurst, P. G., Lewthwaite, S. L., & Triggs, C. M. (1997). Field evaluation of sweetpotato cultivars for resistance to Sclerotinia rot. Proceedings of the NZ Plant Protection Conference , 50 , 89-92. http://www.nzpps.org/journal/50/nzpp_500890.pdf Chitrampalam, P., Turini, T. A., Matheron, M. E., & Pryor, B. M. (2010). Effect of sclerotium density and irrigation on disease incidence and on efficacy of Coniothyrium minitans in suppressing lettuce drop caused by Sclerotinia sclerotiorum . Plant Disease , 94 (9), 1118-1124. https://doi.org/10.1094/pdis-94-9-1118 Dandurand, L. M., Mosher, R. D., & Knudsen, G. R. (2000). Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens. Canadian Journal of Microbiology , 46 (11), 1051-1057. https://doi.org/10.1139/w00-087 Dassanayaka, M. P. (2023). Development of an integrated strategy to manage Sclerotinia sclerotiorum. Doctoral Thesis, Lincoln University, New Zealand. https://hdl.handle.net/10182/16588. Dassanayaka, M. P., Casonato, S. G., & Jones, E. E. (2023). In vitro inhibition of Sclerotinia sclerotiorum mycelial growth and reduction of sclerotial viability by the volatile bioactive compounds of Brassicaceae crops. Journal of Applied Microbiology , 134 (12). https://doi.org/10.1093/jambio/lxad289 de Vrije, T., Antoine, N., Buitelaar, R. M., Bruckner, S., Dissevelt, M., Durand, A., Gerlagh, M., Jones, E. E., Lüth, P., Oostra, J., Ravensberg, W. J., Renaud, R., Rinzema, A., Weber, F. J., & Whipps, J. M. (2001). The fungal biocontrol agent Coniothyrium minitans : production by solid-state fermentation, application and marketing. Applied Microbiology and Biotechnology , 56 (1-2), 58-68. https://doi.org/10.1007/s002530100678 Doheny-Adams, T., Redeker, K., Kittipol, V., Bancroft, I., & Hartley, S. E. (2017). Development of an efficient glucosinolate extraction method. Plant Methods , 13 (1), Article 17. https://doi.org/10.1186/s13007-017-0164-8 Duff, J. D., & Firrell, M. C. (2021). Biofumigation: a cover crop option 12 months of the year to manage three soilborne pathogens ailing the Australian vegetable industry. Global Journal of Agricultural Innovation, Research & Development , 8 , 104-116. https://doi.org/doi.org/10.15377/2409-9813.2021.08.8 Gerlagh, M., Goossen-van de Geijn, H. M., Hoogland, A. E., & Vereijken, P. F. G. (2003). Quantitative aspects of infection of Sclerotinia sclerotiorum sclerotia by Coniothyrium minitans – timing of application, concentration and quality of conidial suspension of the mycoparasite. European Journal of Plant Pathology , 109 (5), 489-502. https://doi.org/10.1023/A:1024294002196 Han, Y. C., Zeng, X. G., Guo, C., Zhang, Q. H., Chen, F. Y., Ren, L., Chen, W. D., & Qin, L. (2022). Reproduction response of Colletotrichum fungi under the fungicide stress reveals new aspects of chemical control of fungal diseases. Microbial Biotechnolgy , 15 (2), 431-441. https://doi.org/10.1111/1751-7915.13754 Hoyte, S. M., Elmer, P. A. G., Parry, F. J., Taylor, J. T., & Marsden, R. S. (2007, October 31). Biological suppression of Sclerotinia sclerotiorum in Kiwifruit Proceedings of the 6th International Symposium on Kiwifruit, Rotorua, New Zealand. Huang, H. C., & Erickson, R. S. (2000). Soil treatment with fungal agents for control of apothecia of Sclerotinia sclerotiorum in bean and pea crops. Plant Pathology Bulletin , 9 , 53-58. Huang, H. C., Erickson, R. S., Phillippe, L. M., Mueller, C. A., Sun, S. K., & Huang, J. W. (2006). Control of apothecia of Sclerotinia sclerotiorum by soil amendment with S-H mixture or Perlka® in bean, canola and wheat fields. Soil Biology and Biochemistry , 38 (6), 1348-1352. https://doi.org/10.1016/j.soilbio.2005.10.015 Huang, H. C., & Janzen, H. H. (1991). Control of carpogenic germination of sclerotia of Sclerotinia sclerotiorum by volatile substances from urea. Plant Protection Bulletin, Taiwan , 33 (3), 283-289. ://CABI:19922319543 Huang, H. C., & Kokko, E. G. (1988). Penetration of hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans without the formation of appressoria. Journal of Phytopathology , 123 (2), 133-139. https://doi.org/10.1111/j.1439-0434.1988.tb04460.x Huang, H. C., & Sun, S. K. (1991). Effects of S-H mixture or perlkaTM on carpogenic germination and survival of sclerotia of Sclerotinia sclerotiorum . Soil Biology and Biochemistry , 23 (9), 809-813. https://doi.org/10.1016/0038-0717(91)90090-7 Jones, D., & Watson, D. (1969). Parasitism and lysis by soil fungi of Sclerotinia sclerotiorum (Lib.) de Bary, a phytopathogenic fungus. Nature , 224 (5216), 287-288. https://doi.org/10.1038/224287a0 Jones, E. E., Mead, A., & Whipps, J. M. (2003). Evaluation of different Coniothyrium minitans inoculum sources and application rates on apothecial production and infection of Sclerotinia sclerotiorum sclerotia. Soil Biology and Biochemistry , 35 (3), 409-419. https://doi.org/10.1016/S0038-0717(02)00292-4 Jones, E. E., Mead, A., & Whipps, J. M. (2004). Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum : control of sclerotinia disease in glasshouse lettuce. Plant Pathology , 53 (5), 611-620. https://doi.org/10.1111/j.1365-3059.2004.01071.x Jones, E. E., Rabeendran, N., & Stewart, A. (2014). Biocontrol of Sclerotinia sclerotiorum infection of cabbage by Coniothyrium minitans and Trichoderma spp. Biocontrol Science and Technology , 24 (12), 1363-1382. https://doi.org/10.1080/09583157.2014.940847 Jones, E. E., & Stewart, A. (2000). Selection of mycoparasites of sclerotia of Sclerotinia sclerotiorum isolated from New Zealand soils. New Zealand Journal of Crop and Horticultural Science , 28 (2), 105-114. https://doi.org/10.1080/01140671.2000.9514130 Kirkegaard, J. A., Gardner, P. A., Desmarchelier, & J.M. , A., J.F. (1993, 5-7 October). Biofumigation: using Brassica species to control pests and diseases in horticulture and agriculture. Ninth Australian Research Assembly on Brassicas, Wagga Wagga, NSW. Kirkegaard, J. A., & Sarwar, M. (1998). Biofumigation potential of brassicas I. Variation in glucosinolate profiles of diverse field-grown brassicas. Plant and Soil , 201 (1), 71-89. https://doi.org/10.1023/A:1004364713152 Kirkegaard, J. A., Sarwar, M., & Matthiessen, J. N. (1998). Assessing the biofumigation potential of crucifers. In G. Thomas & A. A. Monteiro (Eds.), Acta Horticulturae . https://doi.org/10.17660/ActaHortic.1998.459.10 Kurt, Ş., Güneş, U., & Soylu, E. M. (2011). In vitro and in vivo antifungal activity of synthetic pure isothiocyanates against Sclerotinia sclerotiorum . Pest Management Science , 67 (7), 869-875. https://doi.org/https://doi.org/10.1002/ps.2126 Larkin, R. P., & Lynch, R. P. (2018). Use and effects of different Brassica and other rotation crops on soilborne diseases and yield of potato. Horticulturae , 4 (4), Article 37. https://doi.org/10.3390/horticulturae4040037 Lewthwaite, S. L., & Wright, P. J. (2005). Sweetpotato vine resistance to Sclerotinia rot. Acta Horticulture , 670 , 63-69. https://doi.org/10.17660/ActaHortic.2005.670.6 Liu, L., Sun, C., Liu, X., He, X., Liu, M., Wu, H., Tang, C., Jin, C., & Zhang, Y. (2016). Effect of calcium cyanamide, ammonium bicarbonate and lime mixture and ammonia water on survival of Ralstonia solanacearum and microbial community. Scientific Reports , 6 (1), 19037. https://doi.org/10.1038/srep19037 McQuilken, M. P., Mitchell, S. J., Budge, S. P., Whipps, J. M., Fenlon, J. S., & Archer, S. A. (1995). Effect of Coniothyrium minitans on sclerotial survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape. Plant Pathology , 44 (5), 883-896. https://doi.org/10.1111/j.1365-3059.1995.tb02748.x Ojaghian, M. R., Jiang, H., Xie, G.-L., Cui, Z., Zhang, J., & Li, B. (2012). In vitro biofumigation of Brassica tissues against potato stem rot caused by Sclerotinia sclerotiorum . The Plant Pathology Journal , 28 , 185-190. https://doi.org/10.5423/PPJ.2012.28.2.185 Peltier, A. J., Bradley, C. A., Chilvers, M. I., Malvick, D. K., Mueller, D. S., Wise, K. A., & Esker, P. D. (2012). Biology, yield loss and control of Sclerotinia stem rot of soybean. Journal of Integrated Pest Management , 3 (2), B1-B7. https://doi.org/10.1603/IPM11033 Pennycook, S. R. (1985). Fungal fruit rots of Actinidia deliciosa (kiwifruit). New Zealand Journal of Experimental Agriculture , 13 (4), 289-299. https://doi.org/10.1080/03015521.1985.10426097 Pennycook, S. R. (1989). Plant diseases recorded in New Zealand. (Vol. 2). Plant disease division, Department of Scientific and Industrial Research, Auckland, New Zealand. Purdy, L. H. (1979). Sclerotinia sclerotiorum : history, diseases and symptomatology, host range, geographic distribution, and impact. Phytopathology , 69 (8), 875-880. https://doi.org/10.1094/Phyto-69-875 Rabeendran, N. (2000). Biological control of Sclerotina diseases of vegetables . Doctoral Thesis, Lincoln University, New Zealand. https://hdl.handle.net/10182/1502 Reddy, P. P. (2013). Biofumigation. In P. P. Reddy (Ed.), Recent advances in crop protection (pp. 37-60). Springer India. https://doi.org/10.1007/978-81-322-0723-8_4 Saharan, G. S., & Mehta, N. (2008). Sclerotinia diseases of crop plants : biology, ecology and disease management . Springer, New York, USA. Serrano-Pérez, P., De Santiago, A., & Rodríguez-Molina, M. d. C. (2021). Biofumigation with pellets of defatted seed meal of Brassica carinata : factors affecting performance against Phytophthora nicotianae in pepper crops. Frontiers in Sustainable Food Systems , 5 , Article 664531. https://doi.org/10.3389/fsufs.2021.664531 Smolinska, U., & Horbowicz, M. (1999). Fungicidal activity of volatiles from selected cruciferous plants against resting propagules of soil-borne fungal pathogens. Journal of Phytopathology , 147 (2), 119-124. https://doi.org/https://doi.org/10.1046/j.1439-0434.1999.147002119.x Smolińska, U., & Kowalska, B. (2018). Biological control of the soil-borne fungal pathogen Sclerotinia sclerotiorum –– a review. Journal of Plant Pathology , 100 (1), 1-12. https://doi.org/10.1007/s42161-018-0023-0 Tremblay, N., Bélec, C., Coulombe, J., & Godin, C. (2005). Evaluation of calcium cyanamide and liming for control of clubroot disease in cauliflower. Crop Protection , 24 (9), 798-803. https://doi.org/10.1016/j.cropro.2004.12.013 Verkley, G., Silva, M., Wicklow, D., & Crous, P. (2004). Paraconiothyrium , a new genus to accommodate the mycoparasite Coniothyrium minitans , anamorphs of Paraphaeosphaeria , and four new species. Studies in Mycology 50 (2), 323-336. Wang, L., Hu, T., Ji, L., & Cao, K. (2007). Inhibitory efficacy of calcium cyanamide on the pathogens of replant diseases in strawberry. Frontiers of Agriculture in China , 1 (2), 183-187. https://doi.org/10.1007/s11703-007-0031-x Warmington, R., & Clarkson, J. P. (2016). Volatiles from biofumigant plants have a direct effect on carpogenic germination of sclerotia and mycelial growth of Sclerotinia sclerotiorum . Plant and Soil , 401 (1), 213-229. https://doi.org/10.1007/s11104-015-2742-8 Willetts, H. J., & Wong, J. A. L. (1980). The biology of Sclerotinia sclerotiorum , S. trifoliorum, and S. minor with emphasis on specific nomenclature. The Botanical Review , 46 (2), 101-165. https://doi.org/10.1007/BF02860868 Wright, P. J. (2011). Control of sclerotinia leaf drop of lettuce . https://lefroyvalley.co.nz/wp-content/uploads/2011/10/Crop-Food-Research.-Control-of-sclerotinia.pdf Wu, B. M., & Subbarao, K. V. (2008). Effects of soil temperature, moisture, and burial depths on carpogenic germination of Sclerotinia sclerotiorum and S. minor . Phytopathology , 98 (10), 1144-1152. https://doi.org/10.1094/PHYTO-98-10-1144 Yang, L., Li, G., Zhang, J., Jiang, D., & Chen, W. (2011). Compatibility of Coniothyrium minitans with compound fertilizer in suppression of Sclerotinia sclerotiorum . Biological Control , 59 (2), 221-227. https://doi.org/10.1016/j.biocontrol.2011.07.002 Zeng, W., Wang, D., Kirk, W., & Hao, J. (2012). Use of Coniothyrium minitans and other microorganisms for reducing Sclerotinia sclerotiorum . Biological Control , 60 (2), 225-232. https://doi.org/10.1016/j.biocontrol.2011.10.009 Tables Table 1 Mean (adjusted for covariate/sclerotial weight of five sclerotia in each replicate) carpogenic germination, number of apothecia per sclerotium and days for the first apothecium to develop from sclerotia of Sclerotinia sclerotiorum in amended potting mix in the equivalent quantities of three different field rates (50, 75 and 100 t/ha) of Brassica juncea ‘Caliente 199’ fresh biomass and unamended controls at the 15 weeks of incubation. The values presented are back-transformed means. Mean values with different letters within a column are significantly different ( P = 0.05) based on Tukey’s HSD test. s.e.d: standard error of difference of means. Field rate of fresh biomass (t/ha) Carpogenic germination of sclerotia (%) Number of apothecia per germinated sclerotia Days to develop the first apothecium 0 85.0 b 1.7 a 65.1 c 50 77.1 b 2.5 a 56.3 ab 75 46.2 a 2.3 a 54.4 a 100 45.4 a 2.4 a 62.8 bc s.e.d 1.0 0.4 2.7 Table 2 Mean carpogenic germination number of apothecia per sclerotium and days for first apothecium to develop from sclerotia of Sclerotinia sclerotiorum (isolate LUPP2650) in potting mix amended with two different field rates (300 and 400 kg/ha) of Perlka â and unamended control and sclerotial viability of buried sclerotia at the 15 weeks of incubation. The values presented in the table are back-transformed means. Mean values with different letters within a column are significantly different ( P = 0.05) based on Tukey’s HSD test. Field rate of Perlka â (kg/ha) Carpogenic germination of sclerotia (%) Number of apothecia per sclerotium Days to development the first apothecium 400 0.0 a nd * nd * 300 19.2 b 2.1 a 95.2 a Control 83.0 c 2.4 a 85.3 a P value <0.001 0.659 0.109 *Not determined as no sclerotial germination. Table 3 Mean carpogenic germination of free surface sclerotia and viability of buried sclerotia of Sclerotinia sclerotiorum (isolate LUPP2650) in potting mix amended with Coniothyrium minitans or Perlka â alone or in a combined application and unamended control at the 15 weeks of incubation. Mean ± standard error of mean. Treatment Carpogenic germination of sclerotia (%) Number of apothecia per sclerotium Days to develop the first apothecium Sclerotial viability (%) Perlka â only 0.0 nd * nd * 95.0 ± 3.3 C. minitans only 0.0 nd * nd * 5.0 ± 3.3 Perlka â and C. minitans 0.0 nd * nd * 2.5 ± 2.5 Control 83.0 ± 4.5 2.4 ± 0.3 60.5 ± 4.1 97.0 ± 3.1 * Not determined as no sclerotial germination. Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 09 Jul, 2025 Reviewers agreed at journal 17 Nov, 2024 Reviewers invited by journal 11 Nov, 2024 Editor invited by journal 31 Oct, 2024 Editor assigned by journal 30 Oct, 2024 First submitted to journal 26 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5339317","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376604551,"identity":"55d23b2c-1a2e-4cda-a594-3909fbf311e5","order_by":0,"name":"Madhavi P. Dassanayaka","email":"","orcid":"","institution":"Lincoln University","correspondingAuthor":false,"prefix":"","firstName":"Madhavi","middleName":"P.","lastName":"Dassanayaka","suffix":""},{"id":376604552,"identity":"04638695-ee14-49ca-b750-f9a0f0dd3c35","order_by":1,"name":"Seona G. Casonato","email":"","orcid":"","institution":"Lincoln University","correspondingAuthor":false,"prefix":"","firstName":"Seona","middleName":"G.","lastName":"Casonato","suffix":""},{"id":376604553,"identity":"6fbcf944-a3aa-40f3-a2a6-25fd3e41c287","order_by":2,"name":"Eirian Jones","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie3QMUvEMBTA8VcC7dIja0rRz/Cm84SjfhVL4KZDBRcHkZNCuqi3dvBDODomBDIVXB0VZ6FSEB08TPVOOIjWUTD/JSnkx0sK4PP9xQiA/FxszQHr9rPfk6DCjgQ9ZI3HCP2ERoN7+XoNEU0L3Y5xtEfLU3EHxxlsUekkSRGhOq+BJJdmkk6RHbJalQiGw/bMTVCHIAcCCN5Oh8SS/IrlgkEoAZX7gjuWqLcPst+2oy+ysES734IkBL2cAimsSCAsMW7C7BS9IRhJqskwObOkqpVg+QWPsf7mj80NeXoUY04Zf2hejk7yeVkY1jxnm3jjfv5qGF//3gWIfzrflfUd8Pl8vn/cO/4xU5g+HPoiAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1879-4537","institution":"Lincoln University","correspondingAuthor":true,"prefix":"","firstName":"Eirian","middleName":"","lastName":"Jones","suffix":""}],"badges":[],"createdAt":"2024-10-27 01:36:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5339317/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5339317/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70151355,"identity":"6c920460-7077-47a1-90d1-5e560d5ed6d5","added_by":"auto","created_at":"2024-11-29 02:28:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3358184,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Stereo microscopic observations (x10) of antagonism of \u003cem\u003eConiothyrium\u0026nbsp;minitans \u003c/em\u003eisolate LUPP418 with developing mycelia and spores oozing out of the pycnidia in droplets (white arrows) on the surface of a \u003cem\u003eSclerotinia\u0026nbsp;sclerotiorum\u003c/em\u003e sclerotium, (b) apothecial development from sclerotia placed in potting mix amended with \u003cem\u003eBrassica\u0026nbsp;juncea \u003c/em\u003e‘Caliente 199’ plant tissue, (c) apothecia produced from sclerotia placed in unamended potting mix with Perlka\u003csup\u003eâ\u003c/sup\u003e (control), (d) \u003cem\u003eS. sclerotiorum\u003c/em\u003e mycelium growing from the bisected viable sclerotia on potato dextrose agar medium, (e) disintegrated and clumped sclerotia recovered from a mesh bag buried in the \u003cem\u003eConiothyrium\u0026nbsp;minitans \u003c/em\u003etreated potting mix, (f) mycelium and pycnidia of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e growing out from non-viable sclerotia cultured on potato dextrose agar medium.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5339317/v1/e0a7c30ba1711b9cbda7dd38.png"},{"id":70151525,"identity":"28172b88-68ad-49d3-a55e-74387890bd08","added_by":"auto","created_at":"2024-11-29 02:36:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5590602,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5339317/v1/5737243e-3af0-49e6-abd5-188f355a5df1.pdf"},{"id":70151356,"identity":"7c0556fe-7d42-491a-a7db-89f178b7551b","added_by":"auto","created_at":"2024-11-29 02:28:57","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":892387,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5339317/v1/3c1a89768296ad9384331ddf.docx"}],"financialInterests":"","formattedTitle":"Suppression of carpogenic germination and viability of Sclerotinia sclerotiorum sclerotia by biofumigation, biological control and Perlka","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eSclerotinia sclerotiorum\u0026nbsp;\u003c/em\u003eis a ubiquitousplant pathogenic fungus causing substantial yield losses in a broad range of hosts around the world (Boland \u0026amp; Hall, 1994; Bolton et al., 2006; Purdy, 1979).\u0026nbsp;The pathogen is recorded to infect more than 400 plant species from 278 genera belonging to 75 families (Boland \u0026amp; Hall, 1994), with infection of susceptible hosts resulting in up to 100%\u0026nbsp;yield loss (Purdy, 1979; Saharan \u0026amp; Mehta, 2008).\u003cem\u003e\u0026nbsp;Sclerotinia sclerotiorum\u0026nbsp;\u003c/em\u003eis a devastating pathogen in a range of plants in New\u0026nbsp;Zealand (Rabeendran, 2000)\u0026nbsp;including\u0026nbsp;lettuce, canola, beans, peas, potatoes, peanuts, carrots, sweet potatoes, kiwifruit and many other vegetable crops\u0026nbsp;(Broadhurst et al., 1997; Hoyte et al., 2007; Lewthwaite \u0026amp; Wright, 2005; Pennycook, 1985, 1989).\u003c/p\u003e\n\u003cp\u003eSclerotia play a crucial role in the disease cycle of \u003cem\u003eS. sclerotiorum\u003c/em\u003e as they act as inocula and survival structures (Bolton et al., 2006; Smolińska \u0026amp; Kowalska, 2018; Willetts \u0026amp; Wong, 1980). Sclerotia can germinate myceliogenically to infect the lower plant parts directly (Bardin \u0026amp; Huang, 2001; Bolton et al., 2006). Alternatively,\u0026nbsp;apothecia produced by carpogenic germination of sclerotia subsequently produce ascospores, which cause infection of aboveground and soil-line plant tissues (Abawi \u0026amp; Grogan, 1979; Bolton et al., 2006). The\u0026nbsp;sclerotia can remain viable in soil for at least 5 years (Adams \u0026amp; Ayers, 1979; Peltier et al., 2012; Wu \u0026amp; Subbarao, 2008) which makes management of the pathogen in the field more challenging (Smolińska \u0026amp; Kowalska, 2018). Conventional control measures such as using fungicides are largely ineffective at reducing carpogenic germination and/or sclerotial viability. Therefore, research to investigate sustainable control options have been pursued.\u0026nbsp;Moreover,\u0026nbsp;environmentally friendly methods are increasingly being sought due to the\u0026nbsp;potential adverse impacts on the environment and the development of resistance in the pathogen population due to the continuous application of chemical control measures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiofumigation is the process of incorporating macerated plant material of Brassicaceae crops into the soil which, through the hydrolysis of glucosinolate compounds (GSLs) in their plant tissues, results in the production and release of isothiocyanates (ITCs) which can suppress\u0026nbsp;soilborne pests and pathogens\u0026nbsp;(Angus et al., 1994; Kirkegaard et al., 1993; Kirkegaard \u0026amp; Sarwar, 1998). The term biofumigation has also been expanded to include other plant species such as \u003cem\u003eAllium\u003c/em\u003e spp. and \u003cem\u003eSorghum\u003c/em\u003e spp. which also produce biotoxic compounds with comparable suppression to that achieved by Brassicaceae plants (Reddy, 2013).\u0026nbsp;Isothiocyanate compounds released in the biofumigation process of \u003cem\u003eBrassica\u003c/em\u003e spp. have been reported to suppress many soilborne fungi including\u0026nbsp;\u003cem\u003eS. sclerotiorum\u0026nbsp;\u003c/em\u003e(Larkin \u0026amp; Lynch, 2018; Ojaghian et al., 2012; Smolinska \u0026amp; Horbowicz, 1999; Warmington \u0026amp; Clarkson, 2016). Research reports also indicate the potential of synthetic ITCs or ITCs liberated from Brassica tissues to suppress the carpogenic germination and/or viability of \u003cem\u003eS. sclerotiorum\u0026nbsp;\u003c/em\u003esclerotia (Dandurand et al., 2000; Dassanayaka et al., 2023; Kurt et al., 2011; Warmington \u0026amp; Clarkson, 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the many identified mycelial and sclerotial antagonists of \u003cem\u003eS. sclerotiorum\u003c/em\u003e, \u003cem\u003eConiothyrium minitans\u003c/em\u003e (reclassified as \u003cem\u003eParaconiothyrium minitans\u003c/em\u003e by Verkley et al. (2004) but referred to as \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ein this paper) is a widely studied sclerotial parasite and is commercially available as a biological fungicide. \u003cem\u003eConiothyrium minitans\u0026nbsp;\u003c/em\u003ehasbeen utilised as a biocontrol agent in many bioassays and field-based studies and has been demonstrated to suppress the carpogenic germination and viability of \u003cem\u003eS. sclerotiorum\u0026nbsp;\u003c/em\u003esclerotia (Gerlagh et al., 2003; Jones et al., 2003; McQuilken et al., 1995; Zeng et al., 2012). However, its incorporation with other control strategies has not been widely investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePerlka® (calcium cyanamide) is a multi-functional fertilizer which\u0026nbsp;is used as a soil amendment. Calcium cyanamide in Perlka\u003csup\u003eâ\u003c/sup\u003e is known to have a direct fungicidal effect as a soil disinfectant, similar to methyl bromide (Bletsos, 2006; Bourbos et al., 1997; Wang et al., 2007) and indirect effects on controlling soilborne pathogens (Huang \u0026amp; Sun, 1991; Liu et al., 2016; Tremblay et al., 2005).\u0026nbsp;Cyanamide in Perlka\u003csup\u003eâ\u003c/sup\u003e has been shown to be effective in suppressing the carpogenic germination of sclerotia of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e (Huang et al., 2006; Huang \u0026amp; Sun, 1991; Warmington \u0026amp; Clarkson, 2016) but its effect on the viability of sclerotia is unclear.\u003c/p\u003e\n\u003cp\u003eAdministering more than one control measure with different modes of action to target the pathogen in an\u0026nbsp;integrated pest management (IPM) approach rather than relying on a single control measure is recommended.\u0026nbsp;To enable an IPM strategy to be designed to provide effective control of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e further information is required on the effectiveness of the aforementioned alternative non-pesticide control measures to suppress inoculum production by this pathogen. Therefore, the current study investigated; (1) the potential of biofumigants produced by \u003cem\u003eBrassica juncea\u0026nbsp;\u003c/em\u003e‘Caliente 199’ (referred to as ‘Caliente 199’ henceforth), biological control by \u003cem\u003eC. minitans,\u0026nbsp;\u003c/em\u003eand Perlka\u003csup\u003eâ\u003c/sup\u003e to reduce carpogenic germination and sclerotial viability, and (2) the effect of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e and Perlka\u003csup\u003eâ\u003c/sup\u003e as a combined application on the carpogenic germination and sclerotial viability of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePreparation of fungal inoculum\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e isolates LUPP479 and LUPP2650 (NCBI accession number OROR876349 and\u0026nbsp;OR855935, respectively)\u0026nbsp;originating from New Zealand were obtained from the Lincoln University Plant Pathology (LUPP) culture collection and selected for the experiments. Isolate\u0026nbsp;LUPP479 was recovered from cauliflower\u0026nbsp;(\u003cem\u003eBrassica oleracea\u003c/em\u003e) in 1996 and originated in Canterbury (South Island, New Zealand) and isolate LUPP2650 which originated in Auckland (North Island, New Zealand) was recovered from lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e) in 2017. Based on the carpogenic germination results of the previous studies (Dassanayaka, 2023), sclerotia of LUPP475 showed greater germination by subjecting to 4°C preconditioning treatment (conditioned sclerotia) whilst sclerotia of LUPP2650 germinated irrespective of preconditioning. Hence, conditioned sclerotia of LUPP479 and LUPP2650 (in order to maintain the same pretreatment conditions) were used for the initial experiment with biofumigants and unconditioned sclerotia of LUPP2650 was used for other experiments.\u0026nbsp;The sclerotia were produced on sterilized wheat grain\u0026nbsp;following the procedure described by Jones et al. (2003).\u0026nbsp;Conical flasks (250 mL) containing 25 g of wheat grain moistened with 50 mL distilled water were sterilized by autoclaving (121°C, 30 min) twice on consecutive days. Sterilized wheat was inoculated with three \u003cem\u003eS. sclerotiorum\u003c/em\u003e colonised PDA (Potato Dextrose Agar, Difco\u003csup\u003eTM\u003c/sup\u003e) discs (Ø 7 mm) cut from the margins of an actively growing 3-day-old fungal culture. Inoculated flasks were incubated for 4 weeks at 20°C in 24 h dark. Conditioned and unconditioned sclerotia were produced by extended incubation of sclerotia at 4°C and 20°C respectively for another 4 weeks in 24 h dark.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSclerotia were harvested by washing the contents of the flask (wheat and sclerotia) with sterile distilled water. Sclerotia of 2-4 mm in diameter were collected by sieving the contents through a 4 mm sieve and collecting them onto a 2 mm sieve. The sclerotia were placed on sterile paper towels and air-dried inside a laminar flow hood overnight. The sclerotia were further sorted manually to select the sclerotia of uniform size, shape and an intact rind before using them for the experiment. A new batch of sclerotia was produced for each experiment and 10 randomly selected sclerotia were cultured on PDA to ascertain their viability based on production of mycelium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vivo\u003c/em\u003e bioassays for suppression of carpogenic germination of sclerotia\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffect of ‘Caliente 199’ biofumigants\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAn \u003cem\u003ein-vivo\u003c/em\u003e bioassay was set up to test the effect of biofumigants released by macerated plant tissue of ‘Caliente 199’ on carpogenic germination of conditioned sclerotia of two \u003cem\u003eS. sclerotiorum\u003c/em\u003e isolates, LUPP479 and LUPP2650 in sterile potting mix. The soil containing potting mix was prepared based on the modification of John Innes compost No. 1 (https://www.gardeningdata.co.uk/soil/john_innes/john_innes.php). The composition of the potting mix contains 0.58 L of loam, 0.25 L of peat, 0.17 L of sand, mixed with 0.6 g of ground limestone, 1.2 g of\u0026nbsp;Osmocote® (38-0-0), 1.2 g of superphosphate and 0.6 g of potassium sulphate (pH 5.8). The fresh batch of potting mix was prepared and\u0026nbsp;sterilized by autoclaving (121°C, 30 min) before setting up the experiment.\u0026nbsp;Whole plant tissues of ‘Caliente 199’ at 50% flowering was tested at three rates equivalent to field biomass rates of 50, 75 and 100 t ha\u003csup\u003e-1\u003c/sup\u003e. The weights of fresh biomass used to achieve these field rates (considering an incorporation depth of 15 cm) were calculated as 3.35 g, 5.02 g and 6.70 g, respectively to treat 100 mL of potting mix. Clear plastic cylindrical containers (250 mL) were filled with 100 mL of potting mix (approximately 94 g) and the required quantity of macerated plant tissue and mixed thoroughly to the total volume of the potting mix in the container. Controls consisted of unamended potting mix. To avoid any loss of volatile biofumigants from the plant material, all the replicates of one treatment were set up together before moving to the next treatment. For each container, five sclerotia were gently pressed into the surface of the potting mix in a grid pattern with the weight of the five sclerotia measured and recorded before placement in the substrate. The moisture of the potting mix was adjusted to 30% (w/w) by adding sterile distilled water and the weight of the container was recorded (without the lid). The containers were sealed by replacing the lid immediately after adding water. After 2 weeks, the screw caps of the containers were replaced with clear polythene wrappers (40\u0026nbsp;mm thickness, Bio-strategy Ltd) for better light penetration.\u0026nbsp;The moisture level was maintained by watering with sterile distilled water every two weeks to bring the weight of the containers back to their original weight. The containers were incubated at 18°C under 14L:10D light regime (Warmington \u0026amp; Clarkson, 2016) within an incubator (Conviron\u003csup\u003eÒ\u003c/sup\u003e model CMP6010). Eight replicates of each treatment and controls were set up in a randomized complete block design (RCBD). The development of stipes and/or apothecia was monitored and recorded every week for up to 15 weeks. After 15 weeks, the ungerminated sclerotia were recovered and surface sterilized with 1:1 (v:v)\u0026nbsp;solution of ethanol: bleach (with 5.3% w/v NaOCl) for 3 min followed by washing with sterile distilled water three times (Jones et al., 2003). Sclerotia were bisected and plated on\u0026nbsp;PDA\u0026nbsp;and assessed for sclerotial viability based on the mycelial germination of sclerotia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffect of Perlka\u003c/em\u003e\u003cem\u003e\u003csup\u003eâ\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA box bioassay was set up to test the effect of\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e (Alzchem) at\u0026nbsp;two different field rates\u0026nbsp;(300 and 400 kg ha\u003csup\u003e-1\u003c/sup\u003e) on the carpogenic germination of unconditioned sclerotia of \u003cem\u003eS.\u003c/em\u003e \u003cem\u003esclerotiorum\u0026nbsp;\u003c/em\u003eisolate LUPP2650.\u0026nbsp;Clear plastic boxes (120 x 120 x 90 mm) were filled with\u0026nbsp;approximately 500 mL (~450 g) of the sterile soil-based potting mix prepared as previously described. The equivalent quantities to achieve the two field rates of Perlka\u003csup\u003eâ\u003c/sup\u003e, 300 and 400 kg ha\u003csup\u003e-1\u003c/sup\u003e, were calculated considering the depth of incorporation (10 cm) for 500 mL of soil at 0.20 g and 0.15 g for each box. Initially, the boxes were partially filled (up to 4 cm) and Terylene mesh bags (\u0026lt;2 mm pore size and 5 x 5 cm in size) containing 5 sclerotia inside each bag and heat sealed, were placed on the surface of the potting mix. The measured quantities of Perlka\u003csup\u003eâ\u003c/sup\u003e were added and mixed with the remaining potting mix (approximately 150 g) and placed on top of the mesh bags containing the sclerotia to fill the boxes. This resulted in the sclerotial bags being buried approximately 2 cm from the top surface level of the potting mix. The potting mix containing no Perlka\u003csup\u003eâ\u003c/sup\u003e was used as the control.\u0026nbsp;Five free individual sclerotia were placed and pressed into the top of the substrate surface. The moisture of the potting mix was adjusted to 30% w/w by adding sterile distilled water, the lid was replaced, and the\u0026nbsp;weight of the box was recorded.\u0026nbsp;The moisture was maintained by watering with\u0026nbsp;sterile distilled water\u0026nbsp;every two weeks to maintain the weight of each box to its original weight. The boxes were incubated at 18ᵒC under a 14L:10D light regime inside an incubator. Eight replicates of each treatment and control were set up in a RCBD. Stipes and apothecial development were monitored every week for up to 15 weeks. After 15 weeks, the sclerotia in the mesh bags were recovered, and tested for their viability as described previously.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffect of C.\u0026nbsp;minitans and Perlka\u003c/em\u003e\u003cem\u003e\u003csup\u003eâ\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA box bioassay was done to test the effect of\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e and \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ewhen applied alone and in combination, on the carpogenic germination of unconditioned sclerotia of \u003cem\u003eS.\u003c/em\u003e \u003cem\u003esclerotiorum\u0026nbsp;\u003c/em\u003eisolate LUPP2650.\u0026nbsp;Clear plastic boxes filled with\u0026nbsp;approximately 500 mL of soil based potting mix (~450 g) were used for the experiment. Based on the initial screening of 10 different \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003eisolates originating from New Zealand for their \u003cem\u003ein vitro\u003c/em\u003e sclerotial parasitism, isolate LUPP418 (isolated from sclerotia of\u0026nbsp;\u003cem\u003eSclerotinia\u0026nbsp;trifoliorum\u003c/em\u003e) which showed the highest sclerotial parasitism (Fig. 1a) was used in this experiment (Table SI1 and Fig. SI1). The conidial suspension of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e (LUPP418) was prepared by adapting the method described by Jones et al. (2014). A 21-day-old culture was flooded with 10 mL of sterile distilled water and the surface mycelium was scraped using a sterile spreader to dislodge the conidia. The resulting conidial suspension was strained through a double layer of sterile Miracloth to remove any mycelial fragments. The spore concentration was determined using a haemocytometer and 750 mL of 10\u003csup\u003e7\u003c/sup\u003espores mL\u003csup\u003e-1\u003c/sup\u003e spore suspension was prepared. In parallel, the spore suspension was diluted to make 10\u003csup\u003e3\u003c/sup\u003e conidia mL\u003csup\u003e-1\u003c/sup\u003e and 100\u0026nbsp;mL of the suspension was plated on PDA to test for spore viability and germinability. The prepared spore suspension was added to 7.5 kg of the sterile potting mix and mixed thoroughly throughout the entire volume of the potting mix in a 30 L clean plastic box resulting in a final spore concentration of\u0026nbsp;10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eg\u003csup\u003e-1\u003c/sup\u003e potting mix. The \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e amended potting mix was used to fill 16 boxes, with 450 g added to each. Of the 16 boxes, 8 were treated additionally with Perlka\u003csup\u003eâ\u003c/sup\u003e by mixing the measured Perlka\u003csup\u003eâ\u003c/sup\u003e equivalent to the field rate of 400 kg ha\u003csup\u003e-1\u003c/sup\u003e to the top 2 cm layer of the potting mix, as explained previously.\u003c/p\u003e\n\u003cp\u003eA further 7.5 kg of the potting mix was treated with 750 mL of sterile distilled water instead of the spore suspension, as above, and 16 boxes were filled with 450 g each. Of the 16 boxes, 8 were additionally treated with Perlka\u003csup\u003eâ\u003c/sup\u003e of the field rate of 400 kg ha\u003csup\u003e-1\u003c/sup\u003e, and the remaining untreated 8 boxes were considered the control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn each box,\u0026nbsp;Terylene mesh bags (\u0026lt;2 mm pore size and 5 x 5 cm in size) containing 5 sclerotia were buried\u0026nbsp;within the top 2 cm of\u0026nbsp;potting mix\u0026nbsp;and five free individual sclerotia were pressed into the surface of the potting mix as described previously.\u0026nbsp;The boxes were incubated at 18ᵒC under a 14L:10D light regime inside an incubator.\u0026nbsp;Eight replicates of each treatment and control were set up in a RCBD. Stipes and apothecial development were monitored every week for up to 15 weeks. After 15 weeks, the sclerotia in the mesh bags were recovered, and tested for their viability on PDA following the similar procedure explained in the previous experiment.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eThe number of germinated sclerotia (sclerotia which produced at least one stipe), the number of apothecia per sclerotium and the time to produce the first apothecium by individual sclerotia, were recorded in all three experiments. The percentage data (% sclerotial germination) were arcsine square root transformed before the analyses. Data were analysed using general analysis of variance (ANOVA) with mean separated using post-hoc Tukey’s HSD tests at \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.05. The total weight of the five free sclerotia placed in each replicate was used as a covariate in the biofumigant experiment. Values are presented as back transformed means. All the analyses were done using GenStat22 software (VSN international, Hemel Hempstead, United Kingdom).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEffect of ‘Caliente 199’ biofumigants\u003c/p\u003e\n\u003cp\u003e\u003ccite\u003eThe sclerotial germination\u0026nbsp;\u003c/cite\u003eof\u003cem\u003e\u0026nbsp;\u003ccite\u003eboth isolates (LUPP479 and LUPP2650) was observed in the potting mix amended with\u0026nbsp;\u003c/cite\u003e\u003c/em\u003e‘Caliente 199’\u003ccite\u003e(Fig. 1b) and unamended controls.\u003c/cite\u003eThe effect of the quantity of plant material was significant (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) but not the isolate (\u003cem\u003eP\u003c/em\u003e=0.973) on the percentage carpogenic germination of sclerotia. The potting mix treated with ‘Caliente 199’ in the quantities equivalent to the field rates of 75 and 100 t ha\u003csup\u003e-1\u003c/sup\u003e resulted in\u0026nbsp;~46% less carpogenic germination of sclerotia and this was significantly less (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) than the unamended control (Table 1). Treating the potting mix with ‘Caliente 199’ at a quantity equivalent to the field rate of 50 t ha\u003csup\u003e-1\u003c/sup\u003e resulted in similar carpogenic germination to the unamended control and the germination was greater by 41% compared with the sclerotia in potting mix treated with quantity equivalent to 100 t ha\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor the sclerotia which had germinated, the mean number of apothecia per sclerotium varied from 1.6 to 2.5 across both isolates irrespective of the quantity of the biofumigant material. The effect of biofumigant released from different field rates of ‘Caliente 199’ plant tissue significantly (\u003cem\u003eP \u0026lt;\u0026nbsp;\u003c/em\u003e0.001)\u0026nbsp;affected the days to development of the first apothecium by sclerotia.\u0026nbsp;The sclerotia in potting mix amended with the quantity equivalent to the field rates of 75\u0026nbsp;t ha\u003csup\u003e-1\u003c/sup\u003e and 50\u0026nbsp;t ha\u003csup\u003e-1\u003c/sup\u003e, produced the first apothecium, 9 - 11 days earlier, compared with the sclerotia in the untreated control.\u0026nbsp;The viability of the non-germinated sclerotia of both isolates recovered from all the biofumigant treatments and unamended controls at the end of the experiment was high, being \u0026nbsp;³91.0% for both.\u003c/p\u003e\n\u003cp\u003eEffect of Perlka\u003csup\u003eâ\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAlthough most of the sclerotia in the Perlka\u003csup\u003eâ\u003c/sup\u003etreated potting mix produced stipe initials, none in the potting mix treated with 400 t ha\u003csup\u003e-1\u003c/sup\u003e and only a few sclerotia in the potting mix treated with 300 t ha\u003csup\u003e-1\u003c/sup\u003e Perlka\u003csup\u003eâ\u003c/sup\u003e developed fully elongated stipes or apothecia. Only sclerotia which developed stipes or apothecia were considered to have carpogenically germinated. Amending the potting mix with Perlka\u003csup\u003eâ\u003c/sup\u003eequivalent to 400 and 300 t ha\u003csup\u003e-1\u003c/sup\u003e resulted in significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) lower carpogenic germination, with 400 t ha\u003csup\u003e-1\u003c/sup\u003e Perlka\u003csup\u003eâ\u003c/sup\u003e completely inhibiting germination and 300 t ha\u003csup\u003e-1\u003c/sup\u003e Perlka\u003csup\u003eâ\u003c/sup\u003e resulting in 79.6% reduction compared with the unamended control which showed 83.0% carpogenic germination \u003ccite\u003e(Table\u0026nbsp;2)\u003c/cite\u003e. The numbers of apothecia per sclerotium produced in the unamended potting mix (Fig. 1c) and potting mix amended with Perlka\u003csup\u003eâ\u003c/sup\u003e at 300 t ha\u003csup\u003e-1\u003c/sup\u003e were 2.4 and 2.1, respectively and did not differ significantly (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.659) from each other. The sclerotia in the amended potting mix amended with Perlka\u003csup\u003eâ\u003c/sup\u003e at 300 t ha\u003csup\u003e-1\u003c/sup\u003e took 10 more days to develop the first apothecium than the unamended control, however they did not differ significantly (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.109) from each other.\u003c/p\u003e\n\u003cp\u003eStipe initials, which is the initial stage of apothecial formation, were observed in the sclerotia recovered from the mesh bags which were buried in the potting mix treated with Perlka\u003csup\u003eâ\u003c/sup\u003e and controls (unamended) indicating sclerotia were viable. Further, sclerotia produced fungal mycelium characteristics of \u003cem\u003eS. sclerotiorum\u003c/em\u003e on PDA confirming their viability \u003ccite\u003e(Fig. 1d)\u003c/cite\u003e\u003cem\u003e.\u003c/em\u003e Perlka\u003csup\u003eâ\u003c/sup\u003e did not reduce the viability of sclerotia, where the viability of buried sclerotia in the treatments and control was\u0026nbsp;³97.5%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEffect of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e and Perlka\u003csup\u003eâ\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eComplete inhibition of carpogenic germination of sclerotia was observed for either \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e or Perlka\u003csup\u003eâ\u003c/sup\u003e applied alone, or a combined application of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e and Perlka\u003csup\u003eâ\u003c/sup\u003e, whilst the sclerotial germination in the unamended control was 83.0\u0026nbsp;±\u0026nbsp;4.5% (Table 3). When recovering the buried sclerotia from the potting mix treated with \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e, it was observed that the sclerotia were disintegrating and clumped together\u003cem\u003e\u0026nbsp;\u003ccite\u003e(Fig. 1e)\u003c/cite\u003e.\u003c/em\u003e The inside tissue (medulla) of most of the sclerotia were degraded leaving only the outer rind. Additionally, at the time of surface sterilising and subsequent culturing on PDA, the sclerotia were noted to be friable. After transferring to PDA, most of the sclerotia failed to develop \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e mycelium. Instead, other bacterial and fungal contaminations were observed growing from the sclerotia indicating non-viability. The development of mycelium and pycnidia of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e on a few sclerotia was observed \u003ccite\u003e(Fig. 1f)\u003c/cite\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe sclerotia buried in the potting mix treated with \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e only and combined with Perlka\u003csup\u003eâ\u003c/sup\u003e, had reduced sclerotial viability, with 5.0% and 2.5%, respectively. A greater percentage of sclerotia (³95%) in the potting mix treated with only Perlka\u003csup\u003eâ\u003c/sup\u003e, and the untreated control showed mycelial growth of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e on PDA indicating they are viable but no infection by \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e was observed for any of the sclerotia.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOf the different treatments assessed to suppress the carpogenic germination of \u003cem\u003eS. sclerotiorum\u003c/em\u003e sclerotia in the \u003cem\u003ein\u0026nbsp;vivo\u003c/em\u003e bioassays of this study, the antagonism by \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e resulted in the greatest suppression of both carpogenic germination and sclerotial viability. Perlka\u003csup\u003eâ\u003c/sup\u003e was successful in reducing the carpogenic germination but sclerotial viability mostly remained unaffected. Biofumigation released from ‘Caliente 199’ plant tissue in contrast resulted in less suppression of carpogenic germination with sclerotia mostly remaining viable.\u003c/p\u003e\n\u003cp\u003eThe higher quantities of ‘Caliente\u0026nbsp;199’ at the field rates (100 and 75 t ha\u003csup\u003e-1\u003c/sup\u003e) inhibited the carpogenic germination of \u003cem\u003eS. sclerotiorum\u0026nbsp;\u003c/em\u003esclerotia by\u0026nbsp;approximately\u0026nbsp;46% compared with the unamended control. However,\u0026nbsp;the quantity equivalent to the field rate of 50 t ha\u003csup\u003e-1\u003c/sup\u003e did not result in a significant reduction in carpogenic germination. In agreement with these results, Warmington and Clarkson (2016)\u0026nbsp;showed that the dried milled plant material of \u003cem\u003eB.\u0026nbsp;juncea\u003c/em\u003e ‘Caliente 99’ incorporated into compost at the field rate of 100\u0026nbsp;t ha\u003csup\u003e-1\u003c/sup\u003e resulted in\u0026nbsp;~45% reduced carpogenic germination compared with the untreated control after 150 days.\u0026nbsp;\u003cem\u003eBrassica\u0026nbsp;juncea\u003c/em\u003e has the GSL profile of 2-propenyl (sinigrin), 2- phenylethyl and benzyl (Kirkegaard et al., 1998). These GSLs produce aliphatic allyl ITC, and aromatic 2- phenylethyl and benzyl ITCs upon the enzymatic breakdown. \u003cem\u003eBrassica\u0026nbsp;juncea\u003c/em\u003e ‘Caliente 199’ used in the present study is a commercial biofumigant mustard blend which has been reported to produce high levels of sinigrin (Doheny-Adams et al., 2017; Duff \u0026amp; Firrell, 2021).\u0026nbsp;Hence, allyl ITC, along with other ITCs, are likely to have inhibitory activity on the carpogenic germination of the sclerotia. The setup of the bioassay was done as a contact phase experiment in which both aliphatic and aromatic ITCs may be active on the sclerotia and reduce carpogenic germination. It was also clear that increasing the quantity of plant material resulted in an increased inhibition, most likely linked to the greater concentration of ITCs released and available to act on the sclerotia in the potting mix but were not enough to achieve a complete inhibition. Dandurand et al. (2000) however demonstrated complete inhibition of carpogenic germination of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e sclerotia in sterile soil amended with defatted seed meal of \u003cem\u003eB.\u0026nbsp;napus\u003c/em\u003e cv. Dwarf Essex. The comparatively higher inhibition of carpogenic germination reported in the study of Dandurand et al. (2000) than in the present study is most likely due to \u003cem\u003eB. napus\u0026nbsp;\u003c/em\u003ehaving a different GSL profile which may be more effective in suppressing the sclerotial germination. In addition, the use of seed meal may also have increased the efficacy of inhibition, where defatting concentrates the GSLs in seeds (Serrano-Pérez et al., 2021). Further, the incorporation of ‘Caliente 199’ tissue of lower field rates resulted in the early development of apothecia compared with the unamended control. This is possibly due to the toxic bioactive compounds weakening the sclerotial rind and leading to the acceleration of apothecial production. Also, early apothecial production may be a stress response against the toxic bioactive compounds produced by ‘Caliente 199’ tissue, which promoted the fungus to reproduce and sporulate. There are no comparable previous studies with \u003cem\u003eS. sclerotiorum\u003c/em\u003e, but induced sporulation in the presence of high concentration of a fungicide (difenoconazole) has been demonstrated in \u003cem\u003eColletotrichum\u003c/em\u003e spp. by Han et al. (2022).\u0026nbsp;The viability of sclerotia in the biofumigant treatments was\u0026nbsp;³90% and indicates that the bioactive compounds produced by ‘Caliente 199’\u0026nbsp;tissue are not effective enough to kill the sclerotia.\u0026nbsp;This further confirms the results of Dassanayaka et al. (2023) where exposure of sclerotia to ‘Caliente 199’ plant tissue in a volatile phase experiment did not result in significant reduction in sclerotial viability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmending the potting mix with Perlka\u003csup\u003eâ\u003c/sup\u003e resulted in 80 - 100% suppression of carpogenic germination but no substantial reduction in the sclerotial viability. These results also concur with those of Warmington and Clarkson (2016) whereby the mean carpogenic germination of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e sclerotia was reduced by 93% when incubated in pasteurized potting mix treated with 400 kg ha\u003csup\u003e-1\u003c/sup\u003e Perlka\u003csup\u003eâ\u003c/sup\u003e, compared with the untreated\u0026nbsp;control. The partial decomposition of calcium cyanamide in\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e results in the release of\u0026nbsp;cyanamide (Huang \u0026amp; Sun, 1991) urea and ammonia (Huang \u0026amp; Janzen, 1991), which are reported to have an inhibitory effect on the carpogenic germination of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e sclerotia. In the current study, although most of the sclerotia formed stipe initials they did not further differentiate into stipes or apothecia. Hence these compounds seem to exert an inhibitory effect on apothecial development rather than directly killing sclerotia. The application rates used in the current study were clearly not effective to kill the sclerotia. Huang and Sun (1991) reported complete inhibition of carpogenic germination of sclerotia of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e in autoclaved soil treated with\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e at the rates of 0.6, 1% and 2% (w/w) and 100% reduction in viability of buried sclerotia at all three application rates. The application rates of\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e in the study of Huang and Sun (1991) were much higher compared to the present study (0.07% and 0.09% w/w) and are likely to account for the greater reduction in the sclerotial viability. Wright (2011) however demonstrated that the viability of \u003cem\u003eS. minor\u003c/em\u003e sclerotia remained high (73% and 94% viable sclerotia) after application of\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e at the rate of 500 kg ha\u003csup\u003e-1\u003c/sup\u003e in two small-scale lettuce field studies. Hence, it is important to understand the efficacy of\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003eto suppress the pathogen at the recommended field rates (300 -500\u0026nbsp;kg ha\u003csup\u003e-1\u003c/sup\u003e, (Alzchem, 2023)\u0026nbsp;under field conditions and warrants further studies.\u003c/p\u003e\n\u003cp\u003eComplete inhibition of carpogenic germination of sclerotia was achieved by treating the potting mix with the conidial suspension of \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e alone, with almost complete reduction in sclerotia viability. \u003cem\u003eConiothyrium\u0026nbsp;minitans\u003c/em\u003e invades sclerotia via enzymatic hydrolysis and mechanical pressure (Huang \u0026amp; Kokko, 1988). The hypha of the antagonist then grows into the sclerotia and degrades the cells in the cortex and medulla though the production of extracellular enzymes like chitinase and\u0026nbsp;b-1,3 glucanase (Jones \u0026amp; Watson, 1969).\u0026nbsp;It was observed in this study that the buried sclerotia parasitized with\u0026nbsp;\u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ebecame soft and disintegrated leaving only the outer rind. The\u0026nbsp;sclerotia parasitised by \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e failed to produce apothecia and subsequently disintegrated. The results of the present study\u0026nbsp;agree with the results of the pot bioassay done by Jones et al. (2014) where no apothecia were produced by sclerotia inoculated with a spore suspension of \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003e(10\u003csup\u003e6\u003c/sup\u003e spores mL\u003csup\u003e-1\u003c/sup\u003e) and placed in pots containing cabbage seedlings over the 20 week experimental period. The results also concur with the study done by Bennett et al. (2005) that showed greater \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e sclerotial mortality of 90.7% in non-sterilized soil treated with \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003e(10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecfu g\u003csup\u003e-1\u003c/sup\u003e of soil) compared with the untreated control (0% sclerotial mortality) after 90 days incubation.\u0026nbsp;Not only does the sclerotial parasitic activity of different \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003eisolates vary,but the \u0026nbsp;efficacy of a \u003cem\u003eC. minitans\u003c/em\u003e isolate to parasitize different\u0026nbsp;\u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e isolates has also been shown to vary (Jones \u0026amp; Stewart, 2000). Further, the efficacy of sclerotial parasitism also depends on the incubating conditions or soil conditions in the field (de Vrije et al., 2001; Jones et al., 2004). Hence, understanding the potential efficacy of the \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003eisolate tested in this study under field conditions is essential to determine the potential for developing a commercial formulation for incorporation in an IPM programme to control the pathogen.\u003c/p\u003e\n\u003cp\u003eApplying \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ein\u0026nbsp;combination with\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e resulted in complete inhibition of carpogenic germination and high sclerotial mortality similar to the level achieved with the application of \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ealone. Hence, it can be concluded that the sclerotial mortality is predominantly due to the antagonistic activity of \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ebut importantly there was\u0026nbsp;no negative effect from\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003eon the sclerotial parasitic activity by \u003cem\u003eC.\u0026nbsp;minitans\u003c/em\u003e. Since both\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e and \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ealone resulted in\u0026nbsp;complete inhibition of carpogenic germination, any synergistic effect could not be assessed. But Yang et al. (2011) demonstrated a synergistic effect from the combined application of \u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003ewith a compound fertilizer containing N: P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e:K\u003csub\u003e2\u003c/sub\u003eO at 15:15:15 in a pot experiment. Whether lower inoculum concentrations of \u003cem\u003eC. minitans\u003c/em\u003e and Perlka\u003csup\u003eâ\u003c/sup\u003e would result in a synergistic effect should be tested in the field, as it might provide a more economic approach to the effective control of \u003cem\u003eS. sclerotiorum\u003c/em\u003e disease in the field.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results of the study further confirmed the previous literature showing the\u0026nbsp;potential to control \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e by the non-pesticide methods tested. Successful suppression of carpogenic germination or control of Sclerotinia disease by\u0026nbsp;\u003cem\u003eC. minitans\u0026nbsp;\u003c/em\u003eapplicationunder field or similar conditions\u0026nbsp;has been reported by several studies (Chitrampalam et al., 2010; Huang \u0026amp; Erickson, 2000; Jones et al., 2014). The field application of Perlka\u003csup\u003eâ\u003c/sup\u003e has also shown promising results in suppressing apothecial production (Huang et al., 2006). Although the biofumigation treatment with ‘Caliente 199’ did not result in appreciable suppression of sclerotial germination, our previous study reported ‘Caliente 199’ to inhibit the mycelial growth of\u0026nbsp;\u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e (Dassanayaka et al., 2023)\u0026nbsp;and therefore may have activity to prevent mycelial infection of plants by\u0026nbsp;\u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e.\u0026nbsp;Additionally, there are other biofumigant crop types which have been reported to suppress carpogenic germination of \u003cem\u003eS. sclerotiorum\u0026nbsp;\u003c/em\u003eincluding \u003cem\u003eRaphanus sativus\u003c/em\u003e (Warmington \u0026amp; Clarkson, 2016), \u003cem\u003eB. campestris\u0026nbsp;\u003c/em\u003e(Ojaghian et al., 2012)\u0026nbsp;and\u0026nbsp;\u003cem\u003eB. napus\u0026nbsp;\u003c/em\u003e(Dandurand et al., 2000) that could be tested in an integrated approach.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConiothyrium minitans\u003c/em\u003e and\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e were equally effective in suppressing carpogenic germination under the tested conditions. Incorporating \u003cem\u003eC.\u003c/em\u003e \u003cem\u003eminitans\u0026nbsp;\u003c/em\u003ealong with\u0026nbsp;Perlka\u003csup\u003eâ\u003c/sup\u003e resulted in substantial sclerotial mortality which was not achieved by the Perlka\u003csup\u003eâ\u003c/sup\u003e alone per se. This indicates the potential of\u0026nbsp;\u003cem\u003eC.\u003c/em\u003e \u003cem\u003eminitans\u003c/em\u003e to reduce the amount of viable inoculum in the soil and its importance in an IPM program to control the pathogen.\u0026nbsp;The synergistic effect could not be assessed however with the concentration used and\u0026nbsp;future studies could assess with lower concentrations of both components for their potential to provide enhanced suppression of sclerotial germination and viability. Also,\u0026nbsp;there is a potential to integrate these non-pesticide applications with biofumigation or even with reduced application of fungicide.\u0026nbsp;The efficacy of any control measure in the field is influenced by many environmental factors and soil conditions. Therefore, further studies are warranted to assess these control measures alone or as integrated applications to control Sclerotinia disease\u0026nbsp;of crop plants\u0026nbsp;under glasshouse and field conditions. The results of the study can be used in the development of an IPM package to achieve sustainable control of \u003cem\u003eS.\u0026nbsp;sclerotiorum\u003c/em\u003e which is an intractable plant pathogenic fungus.\u003c/p\u003e"},{"header":"Statement and Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eFirst author acknowledges a PhD scholarship awarded by the Accelerating Higher Education and Development operation (AHEAD), Sri Lanka. The authors also gratefully\u0026nbsp;acknowledge\u0026nbsp;an internal grant from the Agriculture and Life Sciences Faculty, Lincoln University to support the research. The authors are grateful to Dr Peter Wright (The New Zealand Institute for Plant and Food Research Limited) for contributing isolates. The authors acknowledge Sandy Hammond for her assistance in the laboratory experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eData available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe work was funded by an internal grant from Lincoln University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u0026nbsp;\u003c/strong\u003eNo competing interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eAll the ethical standards have been followed and no human or animal testing was involved in the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Consent for publication was obtained from all co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThere are no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions: Madhavi P. Dassanayaka\u003c/strong\u003e: Conceptualisation, Methodology, Investigation, Formal Analysis, Writing \u0026ndash; Original Draft, Writing - Review and Editing and Visualization. \u003cstrong\u003eSeona G. Casonato\u003c/strong\u003e: Conceptualisation, Methodology, Formal Analysis, Supervision, Writing - Review and Editing, Validation and Visualization. \u003cstrong\u003eE. Eirian Jones\u003c/strong\u003e: Conceptualisation, Methodology, Supervision, Project Administration and Funding Acquisition, Supplying the Resources, Writing - Review and Editing, Validation and Visualisation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbawi, G. S., \u0026amp; Grogan, R. G. (1979). Epidemiology of diseases caused by \u003cem\u003eSclerotinia\u003c/em\u003e species. \u003cem\u003ePhytopathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;69\u003c/em\u003e, 899-904. https://doi.org/10.1094/Phyto-69-899\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAdams, P. B., \u0026amp; Ayers, W. A. (1979). Ecology of \u003cem\u003eSclerotinia\u0026nbsp;\u003c/em\u003especies. \u003cem\u003ePhytopathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;69\u003c/em\u003e, 896-899. https://doi.org/10.1094/Phyto-69-896\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAlzchem. (2023). \u003cem\u003eApplication rate of Perlka\u0026reg;\u003c/em\u003e. Retrieved 7th October from https://www.alzchem.com/en/brands/perlka/application-area/\u003c/li\u003e\n \u003cli\u003eAngus, J. F., Gardner, P. A., Kirkegaard, J. A., \u0026amp; Desmarchelier, J. M. (1994). Biofumigation: isothiocyanates released from brassica roots inhibit growth of the take-all fungus. \u003cem\u003ePlant and Soil\u003c/em\u003e,\u003cem\u003e\u0026nbsp;162\u003c/em\u003e(1), 107-112. https://doi.org/10.1007/BF01416095\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBardin, S. D., \u0026amp; Huang, H. C. (2001). Research on biology and control of Sclerotinia\u003cem\u003e\u0026nbsp;\u003c/em\u003ediseases in Canada. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;23\u003c/em\u003e(1), 88-98. https://doi.org/10.1080/07060660109506914\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBennett, A. J., Leifert, C., \u0026amp; Whipps, J. M. (2005). Effect of combined treatment of pasteurisation and \u003cem\u003eConiothyrium \u0026nbsp;minitans\u003c/em\u003e on sclerotia of \u003cem\u003eSclerotinia\u0026nbsp;\u003c/em\u003e \u003cem\u003esclerotiorum\u003c/em\u003e in soil. \u003cem\u003eEuropean Journal of Plant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;113\u003c/em\u003e(2), 197-209. https://doi.org/10.1007/s10658-005-2331-4\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBletsos, F. A. (2006). Grafting and calcium cyanamide as alternatives to methyl bromide for greenhouse eggplant production. \u003cem\u003eScientia Horticulturae\u003c/em\u003e,\u003cem\u003e\u0026nbsp;107\u003c/em\u003e(4), 325-331. https://doi.org/10.1016/j.scienta.2005.09.003\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBoland, G. J., \u0026amp; Hall, R. (1994). Index of plant hosts of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;16\u003c/em\u003e(2), 93-108. https://doi.org/10.1080/07060669409500766\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBolton, M. D., Thomma, B. P., \u0026amp; Nelson, B. D. (2006). \u003cem\u003eSclerotinia sclerotiorum\u0026nbsp;\u003c/em\u003e(Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. \u003cem\u003eMolecular Plant Pathology 7\u003c/em\u003e(1), 1-16. https://doi.org/10.1111/j.1364-3703.2005.00316.x\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBourbos, V. A., Skoudridakis, M. T., Darakis, G. A., \u0026amp; Koulizakis, M. (1997). Calcium cyanamide and soil solarization for the control of \u003cem\u003eFusarium solani\u003c/em\u003e f.sp. \u003cem\u003ecucurbitae\u003c/em\u003e in greenhouse cucumber. \u003cem\u003eCrop Protection\u003c/em\u003e,\u003cem\u003e\u0026nbsp;16\u003c/em\u003e(4), 383-386. https://doi.org/10.1016/S0261-2194(96)00110-X\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBroadhurst, P. G., Lewthwaite, S. L., \u0026amp; Triggs, C. M. (1997). Field evaluation of sweetpotato cultivars for resistance to \u003cem\u003eSclerotinia\u003c/em\u003e rot. \u003cem\u003eProceedings of the NZ Plant Protection Conference\u003c/em\u003e,\u003cem\u003e\u0026nbsp;50\u003c/em\u003e, 89-92. http://www.nzpps.org/journal/50/nzpp_500890.pdf\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChitrampalam, P., Turini, T. A., Matheron, M. E., \u0026amp; Pryor, B. M. (2010). Effect of sclerotium density and irrigation on disease incidence and on efficacy of \u003cem\u003eConiothyrium minitans\u003c/em\u003e in suppressing lettuce drop caused by \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003ePlant Disease\u003c/em\u003e,\u003cem\u003e\u0026nbsp;94\u003c/em\u003e(9), 1118-1124. https://doi.org/10.1094/pdis-94-9-1118\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDandurand, L. M., Mosher, R. D., \u0026amp; Knudsen, G. R. (2000). Combined effects of \u003cem\u003eBrassica napus\u003c/em\u003e seed meal and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e on two soilborne plant pathogens. \u003cem\u003eCanadian Journal of Microbiology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;46\u003c/em\u003e(11), 1051-1057. https://doi.org/10.1139/w00-087\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDassanayaka, M. P. (2023). \u003cem\u003eDevelopment of an integrated strategy to manage Sclerotinia sclerotiorum.\u003c/em\u003e Doctoral Thesis, Lincoln University, New Zealand.\u0026nbsp;https://hdl.handle.net/10182/16588.\u003c/li\u003e\n \u003cli\u003eDassanayaka, M. P., Casonato, S. G., \u0026amp; Jones, E. E. (2023). \u003cem\u003eIn vitro\u003c/em\u003e inhibition of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e mycelial growth and reduction of sclerotial viability by the volatile bioactive compounds of Brassicaceae crops. \u003cem\u003eJournal of Applied Microbiology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;134\u003c/em\u003e(12). https://doi.org/10.1093/jambio/lxad289\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ede Vrije, T., Antoine, N., Buitelaar, R. M., Bruckner, S., Dissevelt, M., Durand, A., Gerlagh, M., Jones, E. E., L\u0026uuml;th, P., Oostra, J., Ravensberg, W. J., Renaud, R., Rinzema, A., Weber, F. J., \u0026amp; Whipps, J. M. (2001). The fungal biocontrol agent \u003cem\u003eConiothyrium minitans\u003c/em\u003e: production by solid-state fermentation, application and marketing. \u003cem\u003eApplied Microbiology and Biotechnology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;56\u003c/em\u003e(1-2), 58-68. https://doi.org/10.1007/s002530100678\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDoheny-Adams, T., Redeker, K., Kittipol, V., Bancroft, I., \u0026amp; Hartley, S. E. (2017). Development of an efficient glucosinolate extraction method. \u003cem\u003ePlant Methods\u003c/em\u003e,\u003cem\u003e\u0026nbsp;13\u003c/em\u003e(1), Article 17. https://doi.org/10.1186/s13007-017-0164-8\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDuff, J. D., \u0026amp; Firrell, M. C. (2021). Biofumigation: a cover crop option 12 months of the year to manage three soilborne pathogens ailing the Australian vegetable industry. \u003cem\u003eGlobal Journal of Agricultural Innovation, Research \u0026amp; Development\u003c/em\u003e,\u003cem\u003e\u0026nbsp;8\u003c/em\u003e, 104-116. https://doi.org/doi.org/10.15377/2409-9813.2021.08.8\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGerlagh, M., Goossen-van de Geijn, H. M., Hoogland, A. E., \u0026amp; Vereijken, P. F. G. (2003). Quantitative aspects of infection of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e sclerotia by \u003cem\u003eConiothyrium minitans\u003c/em\u003e \u0026ndash; timing of application, concentration and quality of conidial suspension of the mycoparasite. \u003cem\u003eEuropean Journal of Plant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;109\u003c/em\u003e(5), 489-502. https://doi.org/10.1023/A:1024294002196\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHan, Y. C., Zeng, X. G., Guo, C., Zhang, Q. H., Chen, F. Y., Ren, L., Chen, W. D., \u0026amp; Qin, L. (2022). Reproduction response of \u003cem\u003eColletotrichum\u003c/em\u003e fungi under the fungicide stress reveals new aspects of chemical control of fungal diseases. \u003cem\u003eMicrobial Biotechnolgy\u003c/em\u003e,\u003cem\u003e\u0026nbsp;15\u003c/em\u003e(2), 431-441. https://doi.org/10.1111/1751-7915.13754\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHoyte, S. M., Elmer, P. A. G., Parry, F. J., Taylor, J. T., \u0026amp; Marsden, R. S. (2007, October 31). \u003cem\u003eBiological suppression of Sclerotinia sclerotiorum in Kiwifruit\u003c/em\u003e Proceedings of the 6th International Symposium on Kiwifruit, Rotorua, New Zealand.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHuang, H. C., \u0026amp; Erickson, R. S. (2000). Soil treatment with fungal agents for control of apothecia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e in bean and pea crops. \u003cem\u003ePlant Pathology Bulletin\u003c/em\u003e,\u003cem\u003e\u0026nbsp;9\u003c/em\u003e, 53-58.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHuang, H. C., Erickson, R. S., Phillippe, L. M., Mueller, C. A., Sun, S. K., \u0026amp; Huang, J. W. (2006). Control of apothecia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e by soil amendment with S-H mixture or Perlka\u0026reg; in bean, canola and wheat fields. \u003cem\u003eSoil Biology and Biochemistry\u003c/em\u003e,\u003cem\u003e\u0026nbsp;38\u003c/em\u003e(6), 1348-1352. https://doi.org/10.1016/j.soilbio.2005.10.015\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHuang, H. C., \u0026amp; Janzen, H. H. (1991). Control of carpogenic germination of sclerotia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e by volatile substances from urea. \u003cem\u003ePlant Protection Bulletin, Taiwan\u003c/em\u003e,\u003cem\u003e\u0026nbsp;33\u003c/em\u003e(3), 283-289. \u0026lt;Go to ISI\u0026gt;://CABI:19922319543\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHuang, H. C., \u0026amp; Kokko, E. G. (1988). Penetration of hyphae of \u003cem\u003eSclerotinia sclerotiorum\u0026nbsp;\u003c/em\u003eby \u003cem\u003eConiothyrium minitans\u0026nbsp;\u003c/em\u003ewithout the formation of appressoria. \u003cem\u003eJournal of Phytopathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;123\u003c/em\u003e(2), 133-139. https://doi.org/10.1111/j.1439-0434.1988.tb04460.x\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHuang, H. C., \u0026amp; Sun, S. K. (1991). Effects of S-H mixture or perlkaTM on carpogenic germination and survival of sclerotia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003eSoil Biology and Biochemistry\u003c/em\u003e,\u003cem\u003e\u0026nbsp;23\u003c/em\u003e(9), 809-813. https://doi.org/10.1016/0038-0717(91)90090-7\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJones, D., \u0026amp; Watson, D. (1969). Parasitism and lysis by soil fungi of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e (Lib.) de Bary, a phytopathogenic fungus. \u003cem\u003eNature\u003c/em\u003e,\u003cem\u003e\u0026nbsp;224\u003c/em\u003e(5216), 287-288. https://doi.org/10.1038/224287a0\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJones, E. E., Mead, A., \u0026amp; Whipps, J. M. (2003). Evaluation of different\u003cem\u003e\u0026nbsp;Coniothyrium minitans\u003c/em\u003e inoculum sources and application rates on apothecial production and infection of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e sclerotia. \u003cem\u003eSoil Biology and Biochemistry\u003c/em\u003e,\u003cem\u003e\u0026nbsp;35\u003c/em\u003e(3), 409-419. https://doi.org/10.1016/S0038-0717(02)00292-4\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJones, E. E., Mead, A., \u0026amp; Whipps, J. M. (2004). Effect of inoculum type and timing of application of \u003cem\u003eConiothyrium minitans\u003c/em\u003e on \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e: control of sclerotinia disease in glasshouse lettuce. \u003cem\u003ePlant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;53\u003c/em\u003e(5), 611-620. https://doi.org/10.1111/j.1365-3059.2004.01071.x\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJones, E. E., Rabeendran, N., \u0026amp; Stewart, A. (2014). Biocontrol of \u003cem\u003eSclerotinia sclerotiorum\u0026nbsp;\u003c/em\u003einfection of cabbage by \u003cem\u003eConiothyrium minitans\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTrichoderma\u003c/em\u003e spp. \u003cem\u003eBiocontrol Science and Technology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;24\u003c/em\u003e(12), 1363-1382. https://doi.org/10.1080/09583157.2014.940847\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJones, E. E., \u0026amp; Stewart, A. (2000). Selection of mycoparasites of sclerotia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e isolated from New Zealand soils. \u003cem\u003eNew Zealand Journal of Crop and Horticultural Science\u003c/em\u003e,\u003cem\u003e\u0026nbsp;28\u003c/em\u003e(2), 105-114. https://doi.org/10.1080/01140671.2000.9514130\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKirkegaard, J. A., Gardner, P. A., Desmarchelier, \u0026amp; J.M. , A., J.F. (1993, 5-7 October). \u003cem\u003eBiofumigation: using Brassica species to control pests and diseases in horticulture and agriculture.\u003c/em\u003e Ninth Australian Research Assembly on Brassicas, Wagga Wagga, NSW.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKirkegaard, J. A., \u0026amp; Sarwar, M. (1998). Biofumigation potential of brassicas I. Variation in glucosinolate profiles of diverse field-grown brassicas. \u003cem\u003ePlant and Soil\u003c/em\u003e,\u003cem\u003e\u0026nbsp;201\u003c/em\u003e(1), 71-89. https://doi.org/10.1023/A:1004364713152\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKirkegaard, J. A., Sarwar, M., \u0026amp; Matthiessen, J. N. (1998). Assessing the biofumigation potential of crucifers. In G. Thomas \u0026amp; A. A. Monteiro (Eds.), \u003cem\u003eActa Horticulturae\u003c/em\u003e. https://doi.org/10.17660/ActaHortic.1998.459.10\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKurt, Ş., G\u0026uuml;neş, U., \u0026amp; Soylu, E. M. (2011). \u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e antifungal activity of synthetic pure isothiocyanates against \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003ePest Management Science\u003c/em\u003e,\u003cem\u003e\u0026nbsp;67\u003c/em\u003e(7), 869-875. https://doi.org/https://doi.org/10.1002/ps.2126\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLarkin, R. P., \u0026amp; Lynch, R. P. (2018). Use and effects of different \u003cem\u003eBrassica\u003c/em\u003e and other rotation crops on soilborne diseases and yield of potato. \u003cem\u003eHorticulturae\u003c/em\u003e,\u003cem\u003e\u0026nbsp;4\u003c/em\u003e(4), Article 37. https://doi.org/10.3390/horticulturae4040037\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLewthwaite, S. L., \u0026amp; Wright, P. J. (2005). Sweetpotato vine resistance to \u003cem\u003eSclerotinia\u0026nbsp;\u003c/em\u003erot. \u003cem\u003eActa Horticulture\u003c/em\u003e,\u003cem\u003e\u0026nbsp;670\u003c/em\u003e, 63-69. https://doi.org/10.17660/ActaHortic.2005.670.6\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu, L., Sun, C., Liu, X., He, X., Liu, M., Wu, H., Tang, C., Jin, C., \u0026amp; Zhang, Y. (2016). Effect of calcium cyanamide, ammonium bicarbonate and lime mixture and ammonia water on survival of \u003cem\u003eRalstonia solanacearum\u003c/em\u003e and microbial community. \u003cem\u003eScientific Reports\u003c/em\u003e,\u003cem\u003e\u0026nbsp;6\u003c/em\u003e(1), 19037. https://doi.org/10.1038/srep19037\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMcQuilken, M. P., Mitchell, S. J., Budge, S. P., Whipps, J. M., Fenlon, J. S., \u0026amp; Archer, S. A. (1995). Effect of \u003cem\u003eConiothyrium minitans\u003c/em\u003e on sclerotial survival and apothecial production of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e in field-grown oilseed rape. \u003cem\u003ePlant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;44\u003c/em\u003e(5), 883-896. https://doi.org/10.1111/j.1365-3059.1995.tb02748.x\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOjaghian, M. R., Jiang, H., Xie, G.-L., Cui, Z., Zhang, J., \u0026amp; Li, B. (2012). \u003cem\u003eIn vitro\u003c/em\u003e biofumigation of Brassica tissues against potato stem rot caused by \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003eThe Plant Pathology Journal\u003c/em\u003e,\u003cem\u003e\u0026nbsp;28\u003c/em\u003e, 185-190. https://doi.org/10.5423/PPJ.2012.28.2.185\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePeltier, A. J., Bradley, C. A., Chilvers, M. I., Malvick, D. K., Mueller, D. S., Wise, K. A., \u0026amp; Esker, P. D. (2012). Biology, yield loss and control of \u003cem\u003eSclerotinia\u003c/em\u003e stem rot of soybean. \u003cem\u003eJournal of Integrated Pest Management\u003c/em\u003e,\u003cem\u003e\u0026nbsp;3\u003c/em\u003e(2), B1-B7. https://doi.org/10.1603/IPM11033\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePennycook, S. R. (1985). Fungal fruit rots of Actinidia deliciosa (kiwifruit). \u003cem\u003eNew Zealand Journal of Experimental Agriculture\u003c/em\u003e,\u003cem\u003e\u0026nbsp;13\u003c/em\u003e(4), 289-299. https://doi.org/10.1080/03015521.1985.10426097\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePennycook, S. R. (1989). \u003cem\u003ePlant diseases recorded in New Zealand.\u0026nbsp;\u003c/em\u003e(Vol. 2). Plant disease division, Department of Scientific and Industrial Research, Auckland, New Zealand.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePurdy, L. H. (1979). \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e : history, diseases and symptomatology, host range, geographic distribution, and impact. \u003cem\u003ePhytopathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;69\u003c/em\u003e(8), 875-880. https://doi.org/10.1094/Phyto-69-875\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRabeendran, N. (2000). \u003cem\u003eBiological control of Sclerotina diseases of vegetables\u003c/em\u003e. Doctoral Thesis, Lincoln University, New Zealand. https://hdl.handle.net/10182/1502\u003c/li\u003e\n \u003cli\u003eReddy, P. P. (2013). Biofumigation. In P. P. Reddy (Ed.), \u003cem\u003eRecent advances in crop protection\u003c/em\u003e (pp. 37-60). Springer India. https://doi.org/10.1007/978-81-322-0723-8_4\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSaharan, G. S., \u0026amp; Mehta, N. (2008). \u003cem\u003eSclerotinia diseases of crop plants : biology, ecology and disease management\u003c/em\u003e. Springer, New York, USA.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSerrano-P\u0026eacute;rez, P., De Santiago, A., \u0026amp; Rodr\u0026iacute;guez-Molina, M. d. C. (2021). Biofumigation with pellets of defatted seed meal of \u003cem\u003eBrassica carinata\u003c/em\u003e: factors affecting performance against \u003cem\u003ePhytophthora nicotianae\u003c/em\u003e in pepper crops. \u003cem\u003eFrontiers in Sustainable Food Systems\u003c/em\u003e,\u003cem\u003e\u0026nbsp;5\u003c/em\u003e, Article 664531. https://doi.org/10.3389/fsufs.2021.664531\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSmolinska, U., \u0026amp; Horbowicz, M. (1999). Fungicidal activity of volatiles from selected cruciferous plants against resting propagules of soil-borne fungal pathogens. \u003cem\u003eJournal of Phytopathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;147\u003c/em\u003e(2), 119-124. https://doi.org/https://doi.org/10.1046/j.1439-0434.1999.147002119.x\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSmolińska, U., \u0026amp; Kowalska, B. (2018). Biological control of the soil-borne fungal pathogen \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e \u0026ndash;\u0026ndash; a review. \u003cem\u003eJournal of Plant Pathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;100\u003c/em\u003e(1), 1-12. https://doi.org/10.1007/s42161-018-0023-0\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTremblay, N., B\u0026eacute;lec, C., Coulombe, J., \u0026amp; Godin, C. (2005). Evaluation of calcium cyanamide and liming for control of clubroot disease in cauliflower. \u003cem\u003eCrop Protection\u003c/em\u003e,\u003cem\u003e\u0026nbsp;24\u003c/em\u003e(9), 798-803. https://doi.org/10.1016/j.cropro.2004.12.013\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVerkley, G., Silva, M., Wicklow, D., \u0026amp; Crous, P. (2004). \u003cem\u003eParaconiothyrium\u003c/em\u003e, a new genus to accommodate the mycoparasite \u003cem\u003eConiothyrium minitans\u003c/em\u003e, anamorphs of \u003cem\u003eParaphaeosphaeria\u003c/em\u003e, and four new species. \u003cem\u003eStudies in Mycology 50\u003c/em\u003e(2), 323-336.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWang, L., Hu, T., Ji, L., \u0026amp; Cao, K. (2007). Inhibitory efficacy of calcium cyanamide on the pathogens of replant diseases in strawberry. \u003cem\u003eFrontiers of Agriculture in China\u003c/em\u003e,\u003cem\u003e\u0026nbsp;1\u003c/em\u003e(2), 183-187. https://doi.org/10.1007/s11703-007-0031-x\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWarmington, R., \u0026amp; Clarkson, J. P. (2016). Volatiles from biofumigant plants have a direct effect on carpogenic germination of sclerotia and mycelial growth of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003ePlant and Soil\u003c/em\u003e,\u003cem\u003e\u0026nbsp;401\u003c/em\u003e(1), 213-229. https://doi.org/10.1007/s11104-015-2742-8\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWilletts, H. J., \u0026amp; Wong, J. A. L. (1980). The biology of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, \u003cem\u003eS. trifoliorum,\u003c/em\u003e and \u003cem\u003eS. minor\u003c/em\u003e with emphasis on specific nomenclature. \u003cem\u003eThe Botanical Review\u003c/em\u003e,\u003cem\u003e\u0026nbsp;46\u003c/em\u003e(2), 101-165. https://doi.org/10.1007/BF02860868\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWright, P. J. (2011). \u003cem\u003eControl of sclerotinia leaf drop of lettuce\u003c/em\u003e. https://lefroyvalley.co.nz/wp-content/uploads/2011/10/Crop-Food-Research.-Control-of-sclerotinia.pdf\u003c/li\u003e\n \u003cli\u003eWu, B. M., \u0026amp; Subbarao, K. V. (2008). Effects of soil temperature, moisture, and burial depths on carpogenic germination of\u003cem\u003e\u0026nbsp;Sclerotinia sclerotiorum\u003c/em\u003e and \u003cem\u003eS. minor\u003c/em\u003e. \u003cem\u003ePhytopathology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;98\u003c/em\u003e(10), 1144-1152. https://doi.org/10.1094/PHYTO-98-10-1144\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYang, L., Li, G., Zhang, J., Jiang, D., \u0026amp; Chen, W. (2011). Compatibility of \u003cem\u003eConiothyrium minitans\u003c/em\u003e with compound fertilizer in suppression of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003eBiological Control\u003c/em\u003e,\u003cem\u003e\u0026nbsp;59\u003c/em\u003e(2), 221-227. https://doi.org/10.1016/j.biocontrol.2011.07.002\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZeng, W., Wang, D., Kirk, W., \u0026amp; Hao, J. (2012). Use of \u003cem\u003eConiothyrium minitans\u003c/em\u003e and other microorganisms for reducing \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cem\u003eBiological Control\u003c/em\u003e,\u003cem\u003e\u0026nbsp;60\u003c/em\u003e(2), 225-232. https://doi.org/10.1016/j.biocontrol.2011.10.009\u0026nbsp;\u003cbr\u003e \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Mean\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(adjusted for covariate/sclerotial weight of five sclerotia in each replicate) carpogenic germination, number of apothecia per sclerotium and days for the first apothecium to develop from sclerotia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e in amended potting mix in the equivalent quantities of three different field rates (50, 75 and 100 t/ha) of \u003cem\u003eBrassica\u0026nbsp;juncea\u003c/em\u003e \u0026lsquo;Caliente 199\u0026rsquo; fresh biomass and unamended controls at the 15 weeks of incubation.\u0026nbsp;The values presented are back-transformed means.\u0026nbsp;Mean values with different letters within a column are\u0026nbsp;significantly different (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e=\u0026nbsp;0.05) based on Tukey\u0026rsquo;s HSD test. s.e.d: standard error of difference of means.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"566\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.3216%;\"\u003e\n \u003cp\u003eField rate of fresh biomass (t/ha)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.8551%;\"\u003e\n \u003cp\u003eCarpogenic germination of sclerotia (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4417%;\"\u003e\n \u003cp\u003eNumber of apothecia per germinated sclerotia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3816%;\"\u003e\n \u003cp\u003eDays to develop the first apothecium\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.3216%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26.8551%;\"\u003e\n \u003cp\u003e85.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4417%;\"\u003e\n \u003cp\u003e1.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3816%;\"\u003e\n \u003cp\u003e65.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.3216%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.8551%;\"\u003e\n \u003cp\u003e77.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4417%;\"\u003e\n \u003cp\u003e2.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3816%;\"\u003e\n \u003cp\u003e56.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.3216%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.8551%;\"\u003e\n \u003cp\u003e46.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4417%;\"\u003e\n \u003cp\u003e2.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3816%;\"\u003e\n \u003cp\u003e54.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.3216%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26.8551%;\"\u003e\n \u003cp\u003e45.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4417%;\"\u003e\n \u003cp\u003e2.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3816%;\"\u003e\n \u003cp\u003e62.8\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.3216%;\"\u003e\n \u003cp\u003es.e.d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.8551%;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4417%;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3816%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Mean carpogenic germination number of apothecia per sclerotium and days for first apothecium to develop from sclerotia of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e (isolate LUPP2650) in potting mix amended with two different field rates (300 and 400 kg/ha) of Perlka\u003csup\u003e\u0026acirc;\u003c/sup\u003e and unamended control and sclerotial viability of buried sclerotia at the 15 weeks of incubation. The values presented in the table are back-transformed means. Mean values with different letters within a column are significantly different (\u003cem\u003eP\u003c/em\u003e = 0.05) based on Tukey\u0026rsquo;s HSD test.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"516\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8372%;\"\u003e\n \u003cp\u003eField rate of Perlka\u003csup\u003e\u0026acirc;\u003c/sup\u003e(kg/ha)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.124%;\"\u003e\n \u003cp\u003eCarpogenic germination of sclerotia (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.155%;\"\u003e\n \u003cp\u003eNumber of apothecia per sclerotium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.8837%;\"\u003e\n \u003cp\u003eDays to development\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ethe first apothecium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8372%;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 21.124%;\"\u003e\n \u003cp\u003e0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 20.155%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34.8837%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8372%;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 21.124%;\"\u003e\n \u003cp\u003e19.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 20.155%;\"\u003e\n \u003cp\u003e2.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34.8837%;\"\u003e\n \u003cp\u003e95.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8372%;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 21.124%;\"\u003e\n \u003cp\u003e83.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 20.155%;\"\u003e\n \u003cp\u003e2.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34.8837%;\"\u003e\n \u003cp\u003e85.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8372%;\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 21.124%;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 20.155%;\"\u003e\n \u003cp\u003e0.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34.8837%;\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;*Not determined as no sclerotial germination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Mean carpogenic germination of free surface sclerotia and viability of buried sclerotia of \u003cem\u003eSclerotinia\u0026nbsp;sclerotiorum\u003c/em\u003e (isolate LUPP2650) in potting mix amended with \u003cem\u003eConiothyrium\u0026nbsp;minitans\u003c/em\u003e or Perlka\u003csup\u003e\u0026acirc;\u003c/sup\u003e alone or in a combined application and unamended control at the 15 weeks of incubation. Mean \u0026plusmn; standard error of mean.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28.0992%;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003eCarpogenic germination of sclerotia (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5124%;\"\u003e\n \u003cp\u003eNumber of apothecia per sclerotium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0083%;\"\u003e\n \u003cp\u003eDays to develop the first apothecium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003eSclerotial viability (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28.0992%;\"\u003e\n \u003cp\u003ePerlka\u003csup\u003e\u0026acirc;\u003c/sup\u003e only\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5124%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0083%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e95.0\u0026nbsp;\u0026plusmn;\u0026nbsp;3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28.0992%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. minitans\u003c/em\u003e only\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5124%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0083%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e5.0\u0026nbsp;\u0026plusmn;\u0026nbsp;3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28.0992%;\"\u003e\n \u003cp\u003ePerlka\u003csup\u003e\u0026acirc;\u003c/sup\u003e and \u003cem\u003eC. minitans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5124%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0083%;\"\u003e\n \u003cp\u003end\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e2.5\u0026nbsp;\u0026plusmn;\u0026nbsp;2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28.0992%;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e83.0\u0026nbsp;\u0026plusmn;\u0026nbsp;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5124%;\"\u003e\n \u003cp\u003e2.4\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0083%;\"\u003e\n \u003cp\u003e60.5\u0026nbsp;\u0026plusmn;\u0026nbsp;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1901%;\"\u003e\n \u003cp\u003e97.0\u0026nbsp;\u0026plusmn;\u0026nbsp;3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003eNot determined as no sclerotial germination.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Brassica juncea ‘Caliente 199’, Coniothyrium minitans, integrated pest management (IPM), calcium cyanamide.","lastPublishedDoi":"10.21203/rs.3.rs-5339317/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5339317/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSclerotia play an important role in the disease cycle of \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. Traditional control measures do not effectively suppress germination and viability of sclerotia. This study performed a series of \u003cem\u003ein-vivo\u003c/em\u003e bioassays to investigate the effect of biofumigants produced by \u003cem\u003eBrassica juncea\u003c/em\u003e ‘Caliente 199’, biological control by \u003cem\u003eConiothyrium\u0026nbsp;minitans,\u003c/em\u003e and Perlka\u003csup\u003eâ \u003c/sup\u003eon carpogenic germination and sclerotial viability. \u003cem\u003eConiothyrium\u0026nbsp;minitans\u003c/em\u003e alone or in combination with Perlka\u003csup\u003eâ\u003c/sup\u003e resulted in complete inhibition of carpogenic germination and high sclerotial mortality. Perlka\u003csup\u003eâ\u003c/sup\u003e treatment in quantities equivalent to field rates of 400 and 300 kg ha\u003csup\u003e-1\u003c/sup\u003e resulted in 100% and 77% reduction in sclerotial germination, respectively compared with the untreated control, but viability remained high (³97.5%). Treating the potting mix with plant tissue of ‘Caliente 199’ in quantities equivalent to the field rate of 100 and 75 t ha\u003csup\u003e-1\u003c/sup\u003e reduced carpogenic germination by about 46% compared to unamended control but did not result in significant reduction in sclerotial viability. \u003cem\u003eConiothyrium minitans\u003c/em\u003e and Perlka\u003csup\u003eâ\u003c/sup\u003e were effective in suppressing carpogenic germination but the suppression achieved with biofumigation was much lower. \u003cem\u003eConiothyrium\u003c/em\u003e\u0026nbsp;\u003cem\u003eminitans \u003c/em\u003ewith Perlka\u003csup\u003eâ\u003c/sup\u003e resulted in high sclerotial mortality, which was not achieved with the Perlka\u003csup\u003eâ\u003c/sup\u003e only treatment. These results showed that of the treatments tested, \u003cem\u003eC. minitans\u003c/em\u003e able to reduce the carpogenic germination and the viable inoculum. Further studies are needed to assess the effectiveness and potential of integrating with non-pesticide methods to achieve better control of \u003cem\u003eS. sclerotiorum\u003c/em\u003e in the field.\u003c/p\u003e","manuscriptTitle":"Suppression of carpogenic germination and viability of Sclerotinia sclerotiorum sclerotia by biofumigation, biological control and Perlka","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-29 02:28:52","doi":"10.21203/rs.3.rs-5339317/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-07-10T03:51:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-17T08:45:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-11T11:08:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2024-10-31T06:09:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-30T12:31:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2024-10-26T21:35:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"db8a99d7-eb55-4666-a2ef-9707cbaa4923","owner":[],"postedDate":"November 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-18T02:34:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-29 02:28:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5339317","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5339317","identity":"rs-5339317","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}