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Effect of Alternative Disinfection Treatments Against Fungal Canker in Seeds of Pinus radiata Eugenia Iturritxa,* Marie Laure Desprez-Loustau, Ignacio García-Serna, Eneka Quintana, Nebai Mesanza and Jennifer Aitken
Published PublishedJointly Jointlybybythe theAssociation AssociationofofOfficial OfficialSeed SeedAnalysts Analysts and andthe theSociety SocietyofofCommercial CommercialSeed SeedTechnologists Technologists
Effect of Alternative Disinfection Treatments Against Fungal Canker in Seeds of Pinus radiata Eugenia Iturritxa,* Marie Laure Desprez-Loustau, Ignacio García-Serna, Eneka Quintana, Nebai Mesanza and Jennifer Aitken ABSTRACT Seeds and seedlings of Pinus radiata may be seriously damaged by the canker fungi Diplodia pinea and Fusarium circinatum. is study evaluated alternative seed disinfection treatments against these canker fungi. Experiments were conducted to determine both inhibitory and fungicide effects on colony growth of D. pinea and F. circinatum. Treatments included 24 chemicals at four doses of 1, 10, 100 and 1000 µg mL−1, eight essential oils at five dilutions of 10, 25, 50, 75 and 100% v/v, two antagonistic organisms, Trichoderma harzianum and Trichoderma harzianum, from Basque Country plantations, and four thermotherapy treatments of 55 °C for 8, 9, 10 and 11 hours in vitro. Fungicide and fungistatic effects on inhibition of fungal colony growth were evaluated and phytotoxicity of treatments on P. radiata seeds analyzed. Fusarium circinatum and D. pinea responded in similar ways to the tested treatments, although D. pinea was oen inhibited at lower doses of chemical fungicides and higher doses of essential oils. ermotherapy and Trichoderma treatments showed greater effectiveness against the D. pinea strain than the F. circinatum strain. Germination of P. radiata seeds was highly affected by essential oils application, causing a strong inhibitory effect (≥ 92.25% germination inhibition) with most of the tested oils. RESUMEN Semillas y plantas de Pinus radiata pueden ser dañadas por Fusarium circinatum y Diplodia pinea. El principal objetivo de este estudio fue evaluar tratamientos de control alternativos frente a los hongos D. pinea y F. circinatum para su aplicación en la desinfección de semilla. Se llevan a cabo experimentos para determinar ambos efectos, inhibidor y fungicida, en el crecimiento de las colonias de los hongos patógenos. Los compuestos químicos y organismos utilizados incluyen : 24 compuestos químicos (se testan 4 dosis 1, 10, 100 y 1000 µg por ml), 8 aceites esenciales (cinco diluciones: 10, 25, 50, 75 y 100% v/v), dos colonias de organismos antagonistas (dos tratamientos de Trichoderma harzianum comercial y Trichoderma harzianum procedentes de plantaciones del País Vasco) y cuatro tratamientos de termoterapia (testados en un incubador a 55 °C durante 8, 9, 10 y 11 horas) in vitro. Se evalúa el efecto fungicida y fungiestático en la inhibición del crecimiento de las colonias de hongo y se analiza la fitotoxicidad de los tratamiE. Iturritxa, I. Garcia-Serna, E. Quintana and N. Mesanza, Department of Production and Protection of Plants, NEIKER, Granja Modelo - Arkaute. Apdo. 46. 01080, Vitoria-Gasteiz; M.L. Desprez-Loustau, Department of Forest Pathology, INRA, UMR1202 BIOGECO, Rue Edouard Bourlaux, F33140 Villenave d’Ornon, France; J. Aitkien, e Tree Lab, Level 5, 1135 Arawa Street, PO Box 293, Rotorua, New Zealand. *Corresponding author (E-mail:
[email protected]). Received 14 May 2010.
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entos en semillas de P. radiata. F. circinatum y D. pinea respondieron de forma similar a los tratamientos testados aunque D. pinea fue, a menudo, inhibida a dosis más bajas de fungicidas químicos y más altas de aceites esenciales que F. circinatum. Los tratamientos de termoterapia y con Trichoderma mostraron mayor eficacia frente a la cepa de D. pinea que frente a la de F. circinatum. La germinación de las semillas de P. radiata fue altamente afectada por la aplicación de aceites esenciales, causando un efecto inhibidor fuerte en la mayor parte de los aceites testados (≥ 92,25%, porcentaje de inhibición de la germinación). INTRODUCTION
The two fungi, Diplodia pinea (Desm.) J. Kickx f. [=Sphaeropsis sapinea (Fr.) Dyko & B. Sutton] and Fusarium circinatum Nirenberg & O’Donnell (teleomorph= Gibberella circinata), are major damaging agents of pines, especially P. radiata D. Don, both on seedlings and adult trees (Sinclair and Lyon, 2005). Several lines of evidence suggest that F. circinatum may have originated in Mexico, although it was first reported in southeastern United States causing the pitch canker disease (Gordon, 2006). It has since spread to other parts of the world causing serious damage to pine forests and nurseries in locations such as California (Storer et al., 1994; Storer et al., 2002), South Africa (Viljoen et al., 1994), Chile (Wingfield et al., 2002), and Spain (Collar Urquijo, 1995; Dwinell et al., 1998; Dwinell, 1999; Landeras et al., 2005). Fusarium circinatum is considered to be a major threat to plantation forests in Australia and New Zealand (Gadgil et al., 2003; Ganley et al., 2009). Diplodia pinea has long been known in Europe but the first reports of serious damage only appeared in the 1980s (Swart and Wingfield, 1991). e pathogen has caused extensive damage in plantings of both native and exotic conifers throughout the world (Nicholls and Ostry, 1990; Swart and Wingfield, 1991; Blodgett and Stanosz, 1999; Smith et al., 2002a; Vanneste et al., 2002). Evidence of pine seed infection has been described for both pathogen species (Anderson et al., 1984; Barrows-Broaddus and Dwinell, 1985; Fraedrich and Miller 2000; Vujanovic et al., 2000). Transport of contaminated seeds has been considered to be an important mechanism for the dispersal of the pathogens and their introduction into new areas (Giesler et al., 2000; Smith et al., 2002b; Burgess et al., 2005; Ioos et al., 2009). It is highly desirable to prevent pathogen dispersal by seeds (Jeger, 1999). Such preventive measures are expected to provide effective control of disease spread while having a low environmental impact. is could be achieved by the use of synthetic chemicals, natural substances, biological antagonists or thermotherapy applied to seeds in order to reduce or suppress infection, as has been used for other seed transmitted diseases (Delatour, 1978; Nega et al., 2003; Schmitt et al., 2009, Tinivella et al., 2009). Efficacy of benomyl and some other fungicides has been evaluated against D. pinea and F. circinatum (Stanosz and Smith, 1996; Barnett et al., 1999; Stanosz et al., 2001; Carey et al., 2005), including use as seed treatments (Runion and Bruck, 1988; Allen and Eneback, 2002). Benomyl showed some effectiveness but its use is no longer
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allowed in Europe according to Directive 91/414/CEE (Anonymous, 1991). erefore, it is necessary to look for new products and biological control agents. Essential oils extracted from plants have a number of known potent biological activities including fungicidal effects (Mimica-Duvic et al., 2003; Bozin et al., 2006; Dudai, 2008; Ceng et al., 2009). A number of essential oils have been tested against F. circinatum and D. pinea, but not as seed treatments (Vanneste et al., 2002; Lee et al., 2008; Lee et al., 2009). It is well documented that terpene and resin formation in trees provide natural defense mechanisms against both virulent and non-virulent fungi (Harborne, 1991; Napierała-Filipiak et al., 2002). Previous research on the effect of the pine terpenes ∆3-carene and limonene on Diplodia spore germination showed some inhibition (Chou and Zabkiewicz, 2007). e objective of this study was to assess the efficacy of various chemical, biological and physical treatments against D. pinea and F. circinatum in seeds of P. radiata. Treatments were first tested in vitro for their fungicidal and fungistatic effects, then applied to artificially (inoculated) infested seeds, mimicking real and potential conditions, and taking into account possible phytotoxic effects. MATERIALS AND METHODS Fungal and plant material e D. pinea strain [DPV24, morphotype A, vegetative compatibility group (VCG) 6] used in this study was isolated from a naturally infected P. insignis tree, in Balmaseda, Bizkaia, Spain (García-Serna and Iturritxa, 2008; GarcíaSerna et al., 2008). e F. circinatum strain (CECT20759, FPRPV) was isolated from a P. radiata seedling from Gipuzkoa, Spain [VCG A and mating type 2 (Mat-2)], a unique genotype detected in the region (Iturritxa et al., 2011). e isolates were selected according to their wide distribution, VCG diversity and pathogenicity to P. radiata assessed in previous inoculation trials. Fungi were cultured on potato dextrose agar (PDA) (potato extract powder, 4 g L−1; glucose, 20 g L−1; agar, 15 g L−1) (Oxoid, Unipath Ltd., Bedford, UK). Seeds of P. radiata were obtained from a seed orchard (Umbe, 506130/4798814 UTM coordinates, European 1950 Datum) in the Basque Country and proper phytosanitary conditions and germination quality were checked by the Official Seed Service of Control of Plant and Nurseries, the Basque Government. Previously, naturally infected seeds were collected from seven plantations with the highest damage level caused by canker fungi in the Basque Country. e highest percentages of naturally infected seeds were 3.14% for F. circinatum and 12.28% for D. pinea. e fungi were exclusively isolated from seed coats. Taking into account the introduction of pathogens by imported seeds and the possibility of fungal colonization of the inner seed layers, embryo and gametophyte, artificial inoculation was done to obtain infected embryos, gametophytes and seed coats. In vitro screening Chemical products. Twenty-six commercial chemical products (pure or mixtures) were tested in this trial (Table 1). Benomyl was included as a control
Active ingredient(s)
Dimetomorph, 7.5%; mancozeb, 66.7% Dimetomorph, 7.5%; mancozeb, 66.7% Dimetomorph, 7.5%; mancozeb, 66.7% Dimetomorph, 7.5%; mancozeb, 66.7% Fosetyl-Al, 80% Fosetyl-Al, 80% Fosetyl-Al, 80% Fosetyl-Al, 80% Cyproconazole 10% Cyproconazole, 10% Cyproconazole, 10% Cyproconazole, 10% Quinoline, 50% Quinoline, 50% Quinoline, 50% Quinoline, 50% Benomyl, 50% Benomyl, 50% Benomyl, 50% Benomyl, 50% Pyraclostrobin, 5%; metiram, 55% Pyraclostrobin, 5%; metiram, 55%
Commercial fungicide
Acrobat MZ Acrobat MZ Acrobat MZ Acrobat MZ Aliette express Aliette express Aliette express Aliette express Atemi Atemi Atemi Atemi Beltanol-L Beltanol-L Beltanol-L Beltanol-L Benlate Benlate Benlate Benlate Cabriotop Cabriotop
1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100
Dose (mg L−1)
39.24 ± 4.92 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 59.84 ± 3.48 47.02 ± 2.69 Yes Yes No
No No No No No
xxxx Fusarium circinatum xxxx AIP ± SD Lethality†
100.00 ± 0.00 58.46 ± 9.91 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 97.17 ± 0.43 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 40.11 ± 27.88 100.00 ± 0.00 98.02 ± 0.00
No
Yes Yes No
No No No No No
No
xxxxxxx Diplodia pinea xxxxxxx AIP ± SD Lethality†
Table 1. Growth inhibition by commercial fungicides of Diplodia pinea and Fusarium circinatum, expressed as mean area inhibition percentage (AIP) ± SD.
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Active ingredient(s)
Pyraclostrobin, 5%; metiram, 55% Pyraclostrobin, 5%; metiram, 55% Captan, 48.9% Captan, 48.9% Captan, 48.9% Captan, 48.9% Fludioxonil, 2.5%; mefenoxam 1%; p/v Fludioxonil, 2.5%; mefenoxam 1%; p/v Fludioxonil, 2.5%; mefenoxam 1%; p/v Fludioxonil, 2.5%; mefenoxam 1%; p/v Dithianon, 75%; p/v Dithianon, 75%; p/v Dithianon, 75%; p/v Dithianon, 75%; p/v Tebuconazole, 25% Tebuconazole, 25% Tebuconazole, 25% Tebuconazole, 25% Imazalil, 10%; thiabendazole, 14% Imazalil, 10%; thiabendazole, 14% Imazalil, 10%; thiabendazole, 14% Imazalil, 10%; thiabendazole, 14% Flutriafol, 12.5%
Commercial fungicide
Cabriotop Cabriotop Captan Captan Captan Captan Celest-xl 035 FS Celest-xl 035 FS Celest-xl 035 FS Celest-xl 035 FS Delan Delan Delan Delan Folicur EW 250 Folicur EW 250 Folicur EW 250 Folicur EW 250 FruitGard FruitGard FruitGard FruitGard Impact R
Table 1. (Continued)
10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000
Dose (mg L−1)
23.88 ± 2.71 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 97.58 ± 0.62 96.01 ± 1.46 87.17 ± 2.73 62.83 ± 5.94 75.92 ± 6.61 56.24 ± 4.32 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 96.27 ± 0.44 30.23 ± 1.05 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 Yes
No No
No No
xxxx Fusarium circinatum xxxx AIP ± SD Lethality†
49.09 ± 3.26 0.00 ± 0.00 66.54 ± 9.70 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 86.75 ± 0.28 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 96.96 ± 0.18 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00
No
Yes Yes
No No
No No
xxxxxxx Diplodia pinea xxxxxxx AIP ± SD Lethality†
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Impact R Impact R Impact R Lider 5PM Lider 5PM Lider 5PM Lider 5PM Pomarsol forte Pomarsol forte Pomarsol forte Pomarsol forte Prelude 20FS Prelude 20FS Prelude 20FS Prelude 20FS Premis Premis Premis Premis Ridomil GOLD SL Ridomil GOLD SL Ridomil GOLD SL Ridomil GOLD SL Rovral Aquaflo Rovral Aquaflo Rovral Aquaflo Rovral Aquaflo
Flutriafol, 12.5% Flutriafol, 12.5% Flutriafol, 12.5% Triadimenol, 5% Triadimenol, 5% Triadimenol, 5% Triadimenol, 5% iram, 80% iram, 80% iram, 80% iram, 80% Prochloraz copper chloride, 18% Prochloraz copper chloride, 18% Prochloraz copper chloride, 18% Prochloraz copper chloride, 18% Triticonazole, 2.4% Triticonazole, 2.4% Triticonazole, 2.4% Triticonazole, 2.4% Metalaxyl-m, 46.5% Metalaxyl-m, 46.5% Metalaxyl-m, 46.5% Metalaxyl-m, 46.5% Iprodione, 50% Iprodione, 50% Iprodione, 50% Iprodione, 50%
100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 90.04 ± 1.10 55.00 ± 1.57 40.29 ± 1.35 54.62 ± 4.06 88.21 ± 0.77 1.39 ± 2.78 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 97.91 ± 0.78 95.81 ± 1.83 91.40 ± 2.32 75.60 ± 4.54 67.93 ± 2.91 100.00 ± 0.00 100.00 ± 0.00 31.51 ± 5.26 26.34 ± 3.98 72.63 ± 2.97 26.17 ± 32.10 0.00 ± 0.00 0.00 ± 0.00 Yes No
No No
Yes Yes Yes
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 85.94 ± 0.40 83.42 ± 0.32 82.87 ± 0.54 100.00 ± 0.00 85.87 ± 5.24 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 95.34 ± 2.68 100.00 ± 0.00 100.00 ± 0.00 77.96 ± 9.63 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 99.81 ± 0.09 95.37 ± 0.37 76.82 ± 1.83 Yes No
Yes No
No
No No No No
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Active ingredient(s)
Procymidone, 50% Procymidone, 50% Procymidone, 50% Procymidone, 50% Pyraclostrobin, 25%; p/v Pyraclostrobin, 25%; p/v Pyraclostrobin, 25%; p/v Pyraclostrobin, 25%; p/v Myclobutanil, 24% Myclobutanil, 24% Myclobutanil, 24% Myclobutanil, 24% Himexazol, 70% Himexazol, 70% Himexazol, 70% Himexazol, 70% iabendazole, 50% iabendazole, 50% iabendazole, 50% iabendazole, 50% Propiconazole, 25% Propiconazole, 25% Propiconazole, 25%
Commercial fungicide
Salithiex 50WP Salithiex 50WP Salithiex 50WP Salithiex 50WP Signum Signum Signum Signum Systhane Forte Systhane Forte Systhane Forte Systhane Forte Tachigaren 70WP Tachigaren 70WP Tachigaren 70WP Tachigaren 70WP Tecto Tecto Tecto Tecto Tilt 250 EC Tilt 250 EC Tilt 250 EC
Table 1. (Continued)
1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10 1 1000 100 10
Dose (mg L−1)
39.17 ± 7.50 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 35.38 ± 5.29 33.78 ± 6.47 28.05 ± 3.64 0.00 ± 0.00 91.50 ± 1.04 59.25 ± 1.66 0.00 ± 0.00 0.00 ± 0.00 30.93 ± 3.87 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 83.05 ± 0.43 68.18 ± 6.32 55.84 ± 6.36 0.00 ± 0.00 100.00 ± 0.00 90.95 ± 2.80 15.76 ± 3.68 Yes
xxxx Fusarium circinatum xxxx AIP ± SD Lethality†
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 84.79 ± 1.39 78.97 ± 4.29 76.07 ± 3.56 62.54 ± 4.89 100.00 ± 0.00 100.00 ± 0.00 59.96 ± 2.09 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 64.07 ± 1.88 68.43 ± 6.87 55.84 ± 6.36 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 90.11 ± 4.63
Yes No
xxxxxxx Diplodia pinea xxxxxxx AIP ± SD Lethality†
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No
0.00 ± 0.00 98.72 ± 0.40 73.63 ± 4.97 49.13 ± 6.04 44.80 ± 6.08 95.46 ± 0.32 64.64 ± 1.01 0.00 ± 0.00 0.00 ± 0.00
AIP (%) = (A−B) /A × 100
†Lethality
is only analyzed when AIP is 100%.
1 1000 100 10 1 1000 100 10 1 Propiconazole, 25% Carboxin, 20%; thiram, 20%; p/v Carboxin, 20%; thiram, 20%; p/v Carboxin, 20%; thiram, 20%; p/v Carboxin, 20%; thiram, 20%; p/v Zoxamide, 8.33%; mancozeb, 6.67% Zoxamide, 8.33%; mancozeb, 6.67% Zoxamide, 8.33%; mancozeb, 6.67% Zoxamide, 8.33%; mancozeb, 6.67% Tilt 250 EC Vitavax Flow Vitavax Flow Vitavax Flow Vitavax Flow Electis Electis Electis Electis
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treatment for comparison against alternative fungicides despite being banned in Europe, due to its effectiveness as a fungicide. Fungicide effects on radial growth of the two fungi were studied by the ‘poisoned food technique’ (Adams and Wong, 1991). Required doses of each fungicide were mixed with autoclaved and cooled PDA just before pouring into Petri dishes. e two fungi were then sub-cultured in these Petri dishes by adding a 5-mm diameter disc taken from the margins of stock fungi cultures. Four concentrations were tested per studied substance, 1, 10, 100 and 1000 μg mL−1, in addition to controls with no substance added to the medium. Fungal radial growth was assessed aer incubation at 25 ± 2 °C for 14 d. Four replicates were used for each product and concentration. e inhibition caused by the fungicides was calculated as area inhibition percentage (AIP):
31.29 ± 8.29 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
No
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where A and B are the growth surface of the fungus in control and treated plates, respectively. Inhibition doses ID50 and ID100 were defined as the minimum doses that caused 50 and 100% of mycelium growth inhibition on PDA, respectively, and IDLet as the minimum dose causing lethality. Lethal effect, or lethality, was evaluated in cases of 100% AIP, and aer transferring the strain to a PDA Petri dish. A positive lethality of the active ingredient dose was recorded if, aer a period of 2 weeks, there was neither spore germination nor mycelium growth (Strobel et al., 2001). Spearman’s rank correlation analysis was used to test the association among ID50, ID100 and IDLet to determine if there were consistent similarities among treatment doses and pathogens. Essential oils. e studied oils (Table 2) were obtained from Essentialoilsonline.co. uk, Kobashi essential oils (UK), Salus NaturArzneimittel (Germany) and Omamori (Spain). e oils were originally extracted from different plant parts of 14 plant species
Peel
Lemon / Citrus medica var. limonun L.
Wood
Trees; leaves
Tree bark
Leaves
Stems; branches
Seeds
Sandalwood / Santalum album L.
Tea tree / Melaleuca alternifolia (Maidem & Betche) Cheel
Tepezcohuite / Mimosa tenuiflora (Wild) Pair.
Japanese mint / Mentha arvensis L. var. piperascens Holmes
Cinnamon / Cinnamonum zeylanicum (L.) Breyne
Indian Hemp / Cannabis sativa L. var. indica
yme / ymus vulgaris L.
α-pinene, β-pinene, myrcene, transcaryophylene, α-humulene
Eugenol, eugenyl acetate, β-caryophyllene, benzylbenzoate, linalool, butylated hydroxytoluene, hydroxyanisole
Menthol, manthone
Bioflavonoides, taninos
Terpineol, cineol, pinene, terpinenes
Santalol, santyl acetate, santalene
Timol, carvacrol, cineol, linalol
Carvacrol, terpineol, P-cimento
Cineole (or eucalyptol), 1-pinene, and terpineol, 1-limonene, 3,5,-dimethyl-4-6-di-0-methylphloroacetophenone, dipentene, nerolidiol
α-pinene, camphene, β-pinene, sabinene, myrcene, α-terpinene, linalool, β-bisabolene, limonene, trans-α-bergamotene, nerol, neral
α-pinene, limonene, 1,8-cineole, cis-ocimene, trans-ocimene, 3-octanone, camphor, linalool, linalyl acetate, caryophyllene, terpinen-4-ol and lavendulyl acetate
Main chemical constituents α-pinene, camphene, sabinene, β-pinene, D-3carene, myrcene, α-terpinene, terpinolene, linalool, bornyl acetate, cedrol, cadinene
α-thujone, α-pinene, camphene, β-pinene, p-cymene, α-terpinene, linalool, borneol, β-caryophyllene, thymol and carvacrol
Africa and Palmitic acid, oleic acid, linoleic acid, stearic acid, cyclopropenoid acid, Madagascar geranyl acetate, caryophyllene, α-humulene
England
Sri Lanka
India
India
Australia
Flowers/Leaves Spain
Baobab & Ylang Ylang / Adansonia digitata L. Seed extract & Cananga odorata (Lam.) Hook.f.&T.omsom
Flowers; leaves England
Red yme / ymus zigis L. India
Flowers; leaves Bulgaria
Indonesia
Argentina
England
Country of origin France
Oregano / Origanum vulgare L.
Niaouli / Melaleuca quinquenervia (Cav.) Blaque Trees; leaves
Flowers
Extracted plant part(s) Trees; leaves; cones
Lavender / Lavandula angustifolia Miller (= L. officinalis Chaix = L.vera D.C.)
Common name / Scientific name Cypress / Cupressus sempervirens L.
Table 2. Species, extracted plant parts, origin and main constituents of essential oils tested against Diplodia pinea and Fusarium circinatum cultures.
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including Cupressus sempervirens L., Lavandula angustifolia Miller ( = L. officinalis Chaix = L. vera D.C.), Citrus limonium Risso, Melaleuca quinquenervia (Cav.) S. T. Blake, Origanun vulgare L., ymus zigis L., Santalum album L., Melaleuca alterniflora (Maidem & Betche) Cheel, Mimosa tenuiflora (Willd) Poir., Mentha arvensis L. var. piperascens Holmes, Cinnamonum zeilanicum Breine., Cannabis sativa L. subsp. Indica, Adansonia digitata L., Cananga odorata (Lam.) Hook.f.s.&omsom and ymus vulgaris L. e essential oils were either used pure or aer dilution (10, 25, 50, and 75%, v/v) in sweet almond oil extracted from Prunus amygdalus Basch (Guinama, Ref 5066, density 0.911 at 25 °C), due to their insolubility in water. Sweet almond oil was previously tested and shown to have no influence on fungal growth of the studied species. Sterile PDA was poured into Petri dishes and allowed to cool. A 3-mm mycelial fragment was then transferred to the center of the dish, and 500 µL of tested essential oil dilutions added to the center of the dish to cover the transferred mycelium (Reddy Bhaskara et al., 1997; Bishop and Reagan, 1998). Fungal radial growth was assessed aer incubation at 25 ± 2 °C for 14 d. Six replicates were used for each oil and concentration. e AIP caused by the oils was calculated as described above. Biological antagonists. Two different Trichoderma strains were tested against the F. circinatum and D. pinea strains in paired cultures. Trichoderma harzianum treatments consisted of a mixture of commercial strains (TC) sold as R16 Tricofag (Agromed) at 107 UCL mL−1 (De Liñán, 2009), and a native strain isolated in 2004 from a P. radiata plantation in the Basque Country, in Durango (TPV). In each Petri dish, one mycelium disc (5 mm diameter) of T. harzianum (TC or TPV) and one of either D. pinea or F. circinatum were placed at diametrically opposite sides of a circle, approximately 1 cm from the edge (Bell et al., 1982). In one third of the cases, the two discs were inserted at the same time, in another third, Trichoderma was applied one week aer the pathogens, and in the last third, the pathogens were added one week aer the Trichoderma. Controls of the viability of the studied isolates contained subcultured strains pairing with the same strain. Six replicates were used for each Trichoderma treatment. Incubation, fungal growth and inhibition assessment were performed as previously described. e trials were laid out in a randomized complete block design with two factors, fungal pathogenic species and Trichoderma treatments. Analysis of variance was carried out following arcsine transformation of percentages with two fungal pathogenic species and seven Trichoderma treatments, and Tukey’s test (p ≤ 0.05) was used for mean comparisons using SAS 9.1.3 service pack 3 (SAS Institute Inc., Cary, NC, USA). Treatments on inoculated seeds Seeds of P. radiata were extracted by exposing cones to a temperature of 55 °C for 11 h to cause their opening. Seeds were then superficially disinfected by immersion in 20% bleach (5% sodium hypochlorite) for 2 min. Seeds were then put on the surface of PDA plates totally covered by D. pinea and F. circinatum mycelia (aer 5 d of growth) and incubated at 25 °C with 12 h of light per
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day for 5 d. Controls consisted of seeds superficially disinfected and put on PDA plates without any fungal culture. Chemical, biological and thermotherapy treatments were then applied. Based on in vitro test results, 13 substances with high efficacy were selected for use in the seed trial (total of 24 dilutions). Chemical and biological treatments were applied by immersing and shaking the seeds for 12 h at room temperature in selected dilutions of chemical fungicides and essential oils. Trichoderma treatments were applied in the same way by using a spore suspension of 5 × 108 conidia g−1 of seed. Controls were established by immersion in water instead of chemicals and Trichoderma, and in almond essential oils instead of essential oil treatments. ermotherapy treatments consisted of exposure to 55 °C for 8, 9, 10 and 11 h. For each treatment and fungal species, 60 seeds were distributed in 10-mL vials. Aer treatment, seeds were dissected into separate fractions consisting of seed coat, gametophyte and embryo, following the protocol described by Aitken and Iturritxa (2004) and ISTA (2004), and incubated at 25 ± 2 °C, 12 h light per 24 h, for a period of 14 d. A two-tailed t-test was used to examine infection differences among seed coats, gametophytes and embryos. e effect of treatments on seed germination was evaluated by placing treated seeds on wet filter paper at 20 °C for 28 d (ISTA, 2004). Four hundred seeds were used per treatment and control, with 100 seeds per replication, laid out in a randomized complete block design with four blocks. Treatment effects on germination percentage were subjected to analysis of variance and means separated by Tukey’s test (p ≤ 0.05) RESULTS In vitro screening Chemical products. e inhibitory and lethal effects of the tested fungicides at various doses are summarized in Tables 1 and 3. Fusarium circinatum and D. pinea responded in roughly similar ways to the chemical tested. Spearman’s correlation coefficient for general inhibitory effects of fungicides (obtained by ranking them first for increasing IDLet, followed by ID100 and then ID50) was highly significant (r = 0.81, p < 0.01), and no major discrepancies were detected. However, D. pinea was oen inhibited at lower doses than F. circinatum by many chemicals such as thiabendazole, dithianon and pyraclostrobin. A wide range of effects was observed among fungicides, with at one extreme flutriafol being 100% inhibitory (and even lethal for F. circinatum) at the lowest dose tested (1 μg mL−1), whereas several chemicals showed no inhibitory effect even at their highest dose (1000 μg mL−1), such as fosetyl-Al and captan. Together with benomyl, two other fungicides, metalaxyl-m and propiconazole, showed a lethal effect on both fungi at 1000 μg mL−1 or lower. Flutriafol, tebuconazole and prochloraz copper chloride were also highly effective against the two species. Essential oils. e inhibitory and lethal effects of tested essential oils at various doses are shown in Tables 4 and 5. Fusarium circinatum and D. pinea reacted in similar ways to the essential oils tested, and correlation of general inhibitory effect of essential oils (obtained by ranking them first for increasing IDLet , followed by ID100 and then ID50) was highly significant (r = 0.98, p < 0.01).
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Table 3. Fungicidal activity of chemical fungicides expressed as minimum doses (μg mL−1) for which 50% growth inhibition (ID50), 100% growth inhibition (ID100) and lethality (IDLet) were observed in Diplodia pinea and Fusarium circinatum cultures. xiFusarium circinatumix Active ingredient
Flutriafol
xxxx Diplodia pinea xxxx
ID50
ID100
IDLet
ID50
ID100
IDLet
1
1
1
1
1
>1000
Benomyl
10
10
100
10
10
100
Metalaxyl-m
100
100
1000
100
100
1000
iabendazole
100
1000
1000
10
100
1000
iram
1
10
>1000
1
10
1000
Dithianon
10
100
>1000
1
10
>1000
Tebuconazole
100
100
>1000
100
100
100
Piraclostrobin
100
1000
>1000
10
100
>1000
Cyproconazole
1000
1000
>1000
1000
1000
>1000
1
>1000
>1000
10
100
>1000
Captan Prochloraz
1
>1000
>1000
10
100
>1000
Himexazol
10
>1000
>1000
10
>1000
>1000
Pyraclostrobin + metiram+ elictis
100
>1000
>1000
100
1000
>1000
Propiconazole
100
>1000
>1000
1000
1000
>1000
Carboxim + thiram
100
>1000
>1000
1000
1000
>1000
Fludioxonil + mefenoxam
100
>1000
>1000
>1000
>1000
>1000
Triadimenol
1000
>1000
>1000
100
1000
>1000
Dimetamorph + mancozeb
>1000
>1000
>1000
100
1000
>1000
Procymidone
>1000
>1000
>1000
1
>1000
>1000
Zoxamida + mancozeb
>1000
>1000
>1000
1000
>1000
>1000
Fosetyl-Al
>1000
>1000
>1000
>1000
>1000
>1000
Iprodione
>1000
>1000
>1000
>1000
>1000
>1000
Myclobutanil
>1000
>1000
>1000
>1000
>1000
>1000
Inhibition of D. pinea very oen required higher doses than F. circinatum, e.g. with cypress, lavender, lemon, red thyme, sandalwood, tea tree, tepezcohuite and thyme. e most effective oils were oregano, Japanese mint and cinnamon, all being 100% inhibitory at their lowest tested dose (10%) and even lethal for F. circinatum and D. pinea at a dose of 25% of cinnamon and 50% of oregano. Several essential oils such as Indian hemp and baobab-ylang ylang showed no inhibitory effects but were previously reported to have antiseptic properties (Ael, 2001; Grace et al., 2002), or had low effect at high doses (≥ 50%), such as sandalwood and lemon cypress. Red thyme, thyme and tea tree showed a lethal effect on both fungi at doses of 75% and higher.
Essential Dose oil † (%, v/v) Cypress 10 Cypress 25 Cypress 50 Cypress 75 Cypress 100 Lavender 10 Lavender 25 Lavender 50 Lavender 75 Lavender 100 Lemon 10 Lemon 25 Lemon 50 Lemon 75 Lemon 100 Niaouli 10 Niaouli 25 Niaouli 50 Niaouli 75 Niaouli 100 Red yme 10 Red yme 25 Red yme 50
No
No No No
No
No No No
AIP ± SD 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 36.63 ± 10.01 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 35.59 ± 4.66 58.31 ± 6.21 No No No
No
No No No
Lethality‡
Lethality‡
AIP ± SD 0.00 ± 0.00 0.00 ± 0.00 63.73 ± 5.42 71.24 ± 2.09 92.08 ± 6.97 57.48 ± 3.16 88.33 ± 2.04 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 67.90 ± 5.35 82.07 ± 6.79 100.00 ± 0.00 0.00 ± 0.00 67.32 ± 4.85 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00
xxxxDiplodia pineaxxxx
x Fusarium circinatum x Essential oil † Tepezcohuite Tepezcohuite Tepezcohuite Tepezcohuite Tepezcohuite Oregano Oregano Oregano Oregano Oregano yme yme yme yme yme Japanese mint Japanese mint Japanese mint Japanese mint Japanese mint Indian Hemp Indian Hemp Indian Hemp
Dose (%, v/v) 10 25 50 75 100 10 25 50 75 100 10 25 50 75 100 10 25 50 75 100 10 25 50 AIP ± SD 0.00 ± 0.00 38.68 ± 4.82 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 76.24 ± 6.08 99.87 ± 0.21 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 No Yes Yes No No No No No
No No No No No Yes Yes Yes
Lethality‡
x Fusarium circinatum x
AIP ± SD Lethality‡ 0.00 ± 0.00 0.00 ± 0.00 77.41 ± 3.01 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes 0.00 ± 0.00 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 No 100.00 ± 0.00 No 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
xxxxDiplodia pineaxxxx
Table 4. Growth inhibition of Diplodia pinea and Fusarium circinatum by tested essential oils, expressed as mean area inhibition percentage (AIP) ± SD.
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Red yme 75 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes Indian Hemp 75 0.00 ± 0.00 Red yme 100 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes Indian Hemp 100 0.00 ± 0.00 Sandalwood 10 0.00 ± 0.00 0.00 ± 0.00 Baobab Ylang-Ylang 10 0.00 ± 0.00 Sandalwood 25 40.80 ± 5.98 0.00 ± 0.00 Baobab Ylang-Ylang 25 0.00 ± 0.00 Sandalwood 50 65.82 ± 3.42 0.00 ± 0.00 Baobab Ylang-Ylang 50 0.00 ± 0.00 Sandalwood 75 67.48 ± 5.25 0.00 ± 0.00 Baobab Ylang-Ylang 75 0.00 ± 0.00 Sandalwood 100 66.23 ± 3.13 53.31 ± 4.08 Baobab Ylang-Ylang 100 0.00 ± 0.00 Tea tree 10 67.90 ± 3.68 0.00 ± 0.00 Cinnamon 10 100.00 ± 0.00 No Tea tree 25 95.00 ± 2.24 71.24 ± 16.83 Cinnamon 25 100.00 ± 0.00 Yes Tea tree 50 100.00 ± 0.00 No 100.00 ± 0.00 No Cinnamon 50 100.00 ± 0.00 Yes Tea tree 75 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes Cinnamon 75 100.00 ± 0.00 Yes Tea tree 100 100.00 ± 0.00 Yes 100.00 ± 0.00 Yes Cinnamon 100 100.00 ± 0.00 Yes † Species from which essential oils are derived are listed. For constituents of extracted essential oils from each species, refer to table 3. ‡ Lethality is only analyzed when AIP is 100%.
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00
No Yes Yes Yes Yes
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Trichoderma strains. Area inhibition was greater for D. pinea than F. circinatum in all the essayed treatments. Diplodia pinea growing against commercial Trichoderma had an AIP superior to that of F. circinatum growing in the same conditions. Importantly, D. pinea and F. circinatum growing with Trichoderma strains had the same antagonistic reaction as to TC and TPV treatments. In addition, when either fungal strain was cultivated a week before inoculation with Trichoderma the AIP was 100%. Analysis of variance (data not shown) and mean separation tests indicated that there were significant differences among area inhibition percentages among treatments in both groups, with greater inhibition of D. pinea. Trichoderma treatments were more effective against both pathogens when the Trichoderma had been previously established in the culture (Fig. 1). Furthermore, there were no significant differences in effectiveness between tested Trichoderma strains. Seed treatments e highest seed infection rate corresponded to the untreated control, and treatments with Trichoderma or bleach were not effective as results were quite similar to those of the control (Table 6). Treatment with Folicur and ymus spp. were the most efficient for seed disinfection of P. radiata. Following thermotherapy treatments of 55 °C for 8 h or longer, 100% of the fungi was eliminated from seed coat, embryo and gametophyte of seeds inoculated with D. pinea and F. circinatum, and all
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Table 5. Fungicidal activity of essential oils expressed as minimum doses (%, v/v) for which 50% growth inhibition (ID50), 100% growth inhibition (ID100) and lethality (ID Let) were observed in Diplodia pinea and Fusarium circinatum cultures. xiFusarium circinatumix Essential Oil
ID50
ID100
IDLet
xxxx Diplodia pinea xxxx ID50
ID100
IDLet
Cinnamon
25
25
25
25
25
25
Oregano
10
10
50
10
10
50
Tea tree
10
50
75
25
50
75
yme
10
50
75
25
50
75
Red thyme
50
50
75
50
75
75
Japanese mint
25
25
>100
25
25
>100
Lavender
10
50
>100
50
50
>100
Niaouli
25
50
>100
50
50
>100
Tepezcohuite
50
50
>100
50
75
>100
Lemon
50
100
>100
100
100
>100
Sandalwood
50
>100
>100
100
>100
>100
Cypress
50
>100
>100
>100
>100
>100
Indian Hemp
>100
>100
>100
>100
>100
>100
Baobab-Ylang Ylang
>100
>100
>100
>100
>100
>100
Figure 1. Percentage growth inhibition of Diplodia pinea and Fusarium circinatum compared to pure cultures following Trichoderma harzianum treatments, assessed in paired cultures. Two strains of Trichoderma (PV and TC) were used. e two discs of a paired culture were either added at the same time or at oneweek intervals, with T. harzianum placed first or second. Means with the same letter are not significantly different (p ≤ 0.05) according to Tukey’s test.
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Table 6. Percentage of seed parts infected with Diplodia pinea and Fusarium circinatum (mean ± SD) following different seed disinfection treatments. xxxxxxxxi Infected seed parts (%) ixxxxxxxx Treatment
Seed Part
Diplodia pinea
Fusarium circinatum
Bleach
Coat Gametophyte Embryo
90.00 ± 20.7 b† 23.33 ± 22.09 b 5.00 ± 4.35 ab
87.5 ± 19.94 b 57.81 ± 21.55 b 16.07 ± 8.76 b
Folicur EW 250
Coat Gametophyte Embryo
0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
0.00 ± 0.00 a 2.27 ± 1.54 a 0.00 ± 0.00 a
ymus spp.
Coat Gametophyte Embryo
0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
Trichoderma
Coat Gametophyte Embryo
91.67 ± 26.16 b 25.00 ± 23.25 b 12.5 ± 5.12 b
93.18 ± 22.61 b 85.6 ± 16.28 c 57.08 ± 31 cd
Control
Coat Gametophyte Embryo
100.00 ± 0.00 b 30.77 ± 23.27 b 19.64 ± 17.48 c
100.00 ± 0 b 96.9 ± 6.55 c 30.61 ± 18.87 c
†Means in each column, for each seed part, followed by the same letter, do not significantly differ (p ≤ 0.05) according to Tukey’s test.
treatments (0, 8, 9, 10 and 11 h thermotherapy at 55 °C) significantly differed from the respective controls (data not shown). Influence of treatments on seed germination Most tested essential oils resulted in complete or almost complete germination inhibition, and germination of all seeds following treatment with essential oils was significantly lower than the control (Table 7). In contrast, germination of seeds treated with chemical substances tended to be slightly lower but most did not significantly differ from the control. ermotherapy treatments did not cause a decrease in seed germination, and none of the treatments significantly differed from the control (Table 8). DISCUSSION Suppressing infection of first-year seedlings in seedbeds, as well as of cones in windbreaks and imported seeds, is important since these represent important sources of inoculum (Palmer et al., 1986; Swart and Wingfield, 1991). In general, F. circinatum and D. pinea responded similarly to the various treatments that were tested. However, D. pinea was oen inhibited at lower doses of chemical fungicides and higher doses of essential oils than F. circinatum. ermotherapy and Trichoderma treatments were more effective against D. pinea than F. circinatum. Fungicides with effectiveness similar to benomyl compared to the control, for both pathogenic species, were triticonazole, tebuconazole, prochloraz copper chloride, propiconazole and flutriafol. e latter compound is the only one with a lethal effect at the lowest dose applied against D. pinea and the only
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Table 7. Percentage germination of Pinus radiata seeds following treatment with different concentrations of essential oils and chemical fungicides. Treatment
Concentration
Germination
75% 100% 75% 75% 100% 100% 100% 50% 10% 50% 100% 25% 100% 50% 100%
0 a† 0a 0a 0a 0a 0a 0a 1.5 ab 2 ab 2 ab 2.5 ab 4.5 ab 6.25 ab 7.75 b 78.75 c
1 ppm 1 ppm 20% 10 ppm 10 ppm 1 ppm 10 ppm 10 ppm 1 ppm
80.25 c 83.5 cd 86.75 de 87.75 de 88.75 de 90.75 ef 90.75 ef 91.25 ef 91.75 ef
(%)
Essential oil
yme yme Tea tree Oregano Oregano Niaouli Cinnamon Cinnamon Cinnamon Red yme Red yme Red yme Japanese mint Japanese mint Lavender
Chemical fungicide
Impact R Prelude Bleach 5 minutes Impact R Atemi Atemi Prelude Folicur Folicur
95.5 ef
Control
† Means followed by the same letter do not significantly differ (p ≤ 0.05) according to Tukey’s test.
Table 8. Percentage germination of Pinus radiata seeds following heat treatments at 55 °C for different time periods. Heat treatment
55 °C
Time
Germination
(h)
(%)
8
72.98 a†
55 °C
9
71.94 a
55 °C
10
72.01 a
55 °C
11
73.52 a
Control
—
72.99 a
† Means followed by the same letter do not significantly differ (p ≤ 0.05) according to Tukey’s test.
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one, at the moment, excluded from the list of phytosanitary products accepted in the European Commission review of 17th December in 2009, according to Directive 91/414/CEE (Anonymous, 1991). Other fungicide combinations with similar preventive effects as benomyl, in in vitro bioassays against F. circinatum were benzimidazole/carboxin/thiram and triadimenol/imazalil (Dick and Turner, 1999). Benomyl was the most effective fungicide against D. pinea and F. circinatum (Palmer et al., 1986; Dick and Turner, 1999). In contrast, our results show that flutriafol was the most effective. In addition, control of forest pathogens by alternative products is important for reduction of fungicide applications and production costs. e essential oils of ymus zigis, T. vulgaris and Melaleuca alterniflora were lethal at doses of 75%, and tea tree and Origanum vulgare resulted in lethality at doses of 50%. Lethality at the lowest doses was reached by cinnamon oil at 25% with a fungistatic effect at doses of 10%. Under the conditions of this study, cinnamon oil was the most effective substance against both pathogens. ese results are in agreement with the antifungal effect of cinnamon on Fusarium spp. obtained by Win et al. (2007) and against sapstain fungi by Vanneste et al. (2002). A fungistatic effect is observed at lower doses of 25% for tea tree, Mentha arvensis var. piperascens, at 50% in the cases of Lavanda officinalis, ymus vulgaris, Melaleuca quinquenervia, and at 75% for Mimosa tenuiflora. In general, the doses needed to trigger a fungicidal and fungistatic response are relatively high (lethal doses of ymus zigis, ymus vulgaris, and Melaleuca alterniflora were 75%, and tea tree and oregano lethality was at 50%), and cinnamon showed the most promising lethal activity at 25%. e limiting factor was their generalized inhibition of seed germination. Volatile monoterpenes have been shown to be toxic in the vapor phase toward vascular plants and seeds at high doses (Ward and Nagy, 1966; Owen, 1968). erefore, most of the oils would not be suitable seed treatments at the concentrations tested because of their inhibitory effect on germination and the high concentration required against the studied diseases. Trichoderma spp. are common rhizosphere organisms that grow rapidly and can tolerate a range of environmental conditions, making them good candidates as biocontrol agents (Howell, 2003). In addition, there is evidence that some Trichoderma isolates produce metabolites that enhance plant disease resistance via activation of the host defense response (Hanson and Howell, 2004). In this regard, growth inhibition by Trichoderma strains is greater against D. pinea than F. circinatum. ere was no evidence of a clearly superior AIP caused by the commercial Trichoderma product compared to the native strain. ese results emphasize the need for commercial Trichoderma strains that can be used with P. radiata for control of F. circinatum and D. pinea. Treatments by means of application of Trichoderma are not effective enough for disease disinfection. e observed infection percentages are similar to the controls of untreated seeds and to seeds superficially disinfected with bleach. e treatment was effective in vitro, but when applied to seeds the disinfection was not comparable to the chemical and essential oil treatments. erefore, additional research on seed disinfection is required.
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ermal treatment, under the tested conditions, affected fungal development in both studied species. Exposure to 55 °C for a period of 8 h, together with the application of fungicides, could be a promising combination. No significant differences in the effectiveness and limitations of the various thermal treatments were observed in this study, based on the two tested species. Among the tested treatments, Folicur and thermotherapy were the most effective, raising the possibility of combining thermotherapy with the application of fungicides such as triticonazole, ebuconazole, prochloraz and propiconazole. In the case of ymus zigis, which showed a good disinfection capacity, it is advisable to replace this essential oil by Lavandula officinalis because the latter does not cause an inhibitory effect on germination. Whether these treatments will also be effective for other Pinus species and other fungal diseases remains to be determined. ACKNOWLEDGMENTS We acknowledge and thank Bernardo Samaniego and Arturo Blasco, Seed and Plant Nurseries, Department of Agriculture of the Basque Government, for their support. REFERENCES Adams, P.B. and J.A.L. Wong. 1991. e effect of chemical pesticides on the infection of sclerotia of Sclerotinia minor by the biocontrol agent Sporidesmium sclerotivorum. Phytopathol. 81: 1340–1343. Ael, M. 2001. Essence and alchemy: a natural history of perfume. Gibbs Smith. Aitken, J. and E. Iturritxa. 2004. Disease-free import of Pinus seed. e Tree Lab. 30 September 2004. Report to MAF-For. Biosecurity, New Zealand. Allen, T. and S. Eneback. 2002. Inhibition of four Fusarium species associated with longleaf pine and their effects on seed germination. Auburn Univ. Southern For. Nursery Mgmt. Cooperative. Res. Report 00-1. Anderson, R.C., A.E. Liberta and L.A. Dickman. 1984. Interaction of vascular plants and vesicular-arbuscular mycorrhizal fungi across a soil moisture-nutrient gradient. Oecologia. 64: 111–117. Anonymous. 1991. Directive 91/414/CEE of the council of July 15th, 1991, related to the commercialization of phytosanitary products. Official bulletin no. L 230 of 19/08/ 1991, p. 0001–0032. Barnett J.P., B. Pickens and R. Karrfalt. 1999. Improving longleaf pine seedling establishment in the nursery by reducing seedcoat microorganisms. In Proceedings of the 10th biennial southern silvicultural research conference; 1999 Feb 16–18; Shreveport, LA. Asheville (NC): USDA For. Serv. Southern Res. Station. Gen. Tech. Rep. SRS30. p 339–343. Barrows-Broaddus, J. and L.D. Dwinell. 1985. Branch dieback and cone and seed infection caused by Fusarium moniliforme var. subglutinans in a loblolly pine seed orchard in South Carolina. Phytopathol. 75: 1104–1108. Bell, D.K., H.D. Wells and C.R. Markham. 1982. In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathol. 72: 379–382. Bishop, C.D. and J. Reagan. 1998. Control of the storage pathogen Botrytis cinerea on Dutch white cabbage (Brassica oleracea var. capitata) by the essential oil Melaleuca alterniflora. J. Essent. Oil Res. 10: 57–60.
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