V. Cebolla, P.F. Martinez, A. Del Busto, D. Gómez De Barreda, J.J. Tuset
Instituto Valenciano de Investigaciones Agrarias 46113 Moncada (Valencia) Spain

Artículo Publicado en: Acta Horticulturae 382:156-163 el año 1994

Keywords Soil-fungi, Disinfestation, Solarization, soil solar heating.

An important dosage reduction on MBr fumigation can be achieved by combination with soil solar heating during summer in the Mediterranean coast of Spain. This technique allows a significant action over fungi, weeds, plant health and harvest production, at least at the same level than fumigation with MBr at a normal dosage (about 60g/m2 of soil). Controlled fungi include Phytophthora cactorum, P. parasitica, Pythium spp., Fusarium oxysporum f.sp. dianthi and Rhizoctonia solani. Crops include carnations, tomatoes, carrots and strawberries. Spontaneous flora is also controlled. 1. Introduction Soil solar heating (Solarization) in areas with high Summer insolation presents an alternative method to control a number of soil borne biological agents, with proven efficacy in the control against nematodes, pathogenic fungi, and adventitious weeds (Katan 1976). In the geographical latitude of Valencia the limitations presented by solarization are basically the meteorological unsteadiness which produces uncertain results, and the long period of treatment which does not adapt always to the crop calendar. The possibility of combining solarization with fumigants at very low dosages has been put forward by a number of authors (Cebolla and García, 1984, Malathrakis 1987, Martinez et al. 1987) and their results evaluated both in health and in yield of crops, have been comparable to the conventional treatment with fumigants. A method of fungal management is suggested here, which allows their exact location in the soil, and the subsequent recovery from the exact point in which the inoculum was placed, in order to study the direct effect on the overall structures. In order to check the small dosage techniques for Methyl Bromide (MBr), several crops conducted on naturally infested soils are set to confirm the practical application to the most common cultures in the area. 2. Material and Methods 2.1. Pathogens --------- 2.1.1. Inoculum Phytophthora parasitica, P. cactorum both isolated from strawberry, Pythium spp. and Rhizoctonia solani isolated from melon, and Fusarium oxysporum f.sp. dianthi isolated from carnation were used for preparing artificial inocula. Fungi were grown in PDA Petri dishes until complete colonization. Simultaneously, small balls of expanded clay (ARLITA) with a size between 6 and 10 mm diam., which are extremely porous, are autoclave sterilized in a nutrient broth consistent in potato extract plus 40g/l glucose, in order to introduce nutrient medium into the ball cavities. Upon exit from autoclave the air contained in the cavities of the expanded clay has been substituted by the nutrient medium. Subsequently, the balls were transferred to the Petri dishes colonized by fungi, and were incubated until micellia covers completely the surface and cavities of the balls. In such a way the fungal structures were placed inside the clay ball cavities. Mesh bags containing 20 balls were attached by fastening them to rigid plastic mesh pieces ensuring correct separation between three different depths, 4, 8 and 24 cm. under soil surface. After treatment, the bag contents are recovered by unearthing carefully, and transferring to the following selective media, Ponchet for Pythium and Phytophthora (Ponchet 1972), Komada for Fusarium (Komada 1975), SEIA for Rhizoctonia (Menzies 1967). A record is made of the number of balls within which the fungi has succeeded to survive after the treatment, and grow in the medium. 2.1.2. Treatments Normal fumigation with MBr (2% Chloropicrine) at a dosage of 56g/m2 (56) was compared with Solarization (Mulch), consisting in mulching with standard PE 50 micron thick, Solarization under plastic Tunnel (Tu) plus mulching, mulching plus 14g/m2 MBr (14A), mulching plus 28 g/m2 MBr (28 A), Tunnel plus mulching plus 14 g/m2 MBr (14T), uncovered soil was left as control (Check). Experimental plots (6 x 3 m) were prepared with the ground in season, by cross tilling with a cultivator followed by another with rotovator. Application of treatments was done at the end of July. The plastic sheets were left on place during 15 and 30 days for the treatments (Mulch),(Tu),(14A),(14T) and (28 A) in order to study the effect of exposure time to the sun radiation. For all these treatments the plots were irrigated under the plastic sheet one day after application of MBr. The experimental design was completely randomized with two replicates of each treatment and exposure time. Test was repeated during two years. Comparison of results was made through the Duncan multiple range test. 2.1.3. Weeds The effect of treatments, previously described, on weeds was studied by visual observation of spontaneous flora development, and comparison with control. In all instances soil samples were collected and incubated under optimal conditions in greenhouse to study the effects on seed of adventitious weed (Carretero 1977). 2.1.4 Temperatures Soil temperatures for Control, Mulching and Tunnel were measured continuously at depths of 4, 8, 16, 32, and 64 cm by means of platinum probes Pt-100, calibrated and connected to a data logger. 2.2. Crops ----- The experiments were set in different sites along the Valencian coast. In all the experiments MBr was applied using the cold technique, at the dosages specified for each crop, under a plastic PE sheet that remained covering the soil at least for 1 day. On the treatments of Solarization + MBr, the soil was covered with a PE plastic sheet, then the gas was dosed under the plastic, and 1 day afterward the soil was irrigated under the plastic sheet. PE sheet remained the period specified in each paragraph. 2.2.1. Strawberries The experiment was set in a soil from which Verticillium dahliae was isolated from artichokes on several plants from the previous crop though along the studied crop this fungus was not isolated, including check plots. The phytopathological problem consisted in a soil complex where R. solani was the most frequently isolated pathogen. Experimental design consist in plots of 37m2, 4 replicates per treatment. Treatments were control, Fumigation with MBr (33% Chloropicrine) at dosage 70 gr/m2 and a combination of soil solarization with 17,5g/m2 Mbr. Solarization started on end of June and remained up to middle August. Yield was recorded from 5th March to 9th June. 2.2.2. Carrots The experiment was set in a soil naturally infested with Pythium spp. isolated from many plants from the preceding crop. Experimental design consist in plots of 200 m2, 3 replicates per treatment. Treatments were control, Solarization and Solarization + 7g/m2 Mbr. Solarization sheets remained from middle of July up to end August. Yield was recorded at the end of the crop. 2.2.3. Tomatoes The experiment was set in a greenhouse without any special pathogen in the soil, but in which a tomato cv. Cobra F1 crop was repeated along two years. Experimental design consist in plots of 130 m2, 2 replicates per treatment. Treatments were control, Fumigation with 56g/m2 MBr, Solarization and Solarization + 14g/m2 MBr. Solarization started at the beginning of July and remained up to middle of August. Yield was recorded along the crop, from 6th November to 9th January. 2.2.4. Carnations Carnation crop (cv. Lena) was accomplished in big containers (40x40x80 cm) filled with a substrate made with a mixture of sandy soil and 30% peat. The substrate was infested with 2Kg per container of carnation stems infected with F.o. dianthi. Experimental design consistent in 6 replicates per treatment, and 45 cuttings per container, each replicate. Treatments were control non disinfested, Standard fumigation with MBr at 70g/m2, soil solarization, solarization with the addition of MBr at 28g/m2 and solarization with MBr at 14 g/m2. Solarization consisted on mulching under closed greenhouse, starting on 27th July up to September 3rd. A preliminar crop was started on 6th October and a second crop on 2nd July. Flowers were collected and recorded up to 11 July. At the end of the crop plants were cut by the first knot and the necrotic vessel ratio was recorded. 3. Results 3.1. Pathogens --------- As Pathogens concerns, the Duncan test (p=0.05) applied within each fungus, treatment and duration of mulching, does not suggest significant differences in any case between fumigation treatments 56, 28A, 14A and 14T which show the best, results after just 15 exposure days, up to the maximum studied depth (Table 1, 2). Tunnel+Mulching (Tu) proves significantly better than the control in all instances. Differences between (Tu) and fumigant treatments are significant for F. oxysporum and R. solani when studying the values from 0-24cm. On the superficial layer 0-4cm (Tu) proves as efficient as the treatments with fumigants for P. parasitica, P. cactorum and Pythium, but not for F.o. dianthi and R. solani. 3.1.1 Weeds The best results (table 4) are shown by MBr 56g/m2, mulching and mulched tunnel with low dose of MBr, the poorest result was obtained with mulching. The rest of the treatments give intermediate efficacy. From samples incubated in greenhouse (table 5) weeds apear with significant differences just when compared vs. control. Mulching does not show significant differences with fumigation treatments. 3.1.2 Temperatures Temperatures reached under mulching (table 3) are higher than in naked soil at any depth, but the highest increase is shown by mulching under tunnel which arrives to 47 h above 43ºC at a depth of 32cm. 3.2. Crops ----- The addition of small amounts of Mbr to solarization improves its efficacy (table 6) and produce similar crop yields as MBr at normal dosage, while solarization has an intermediate level between the control and the fumigation treatments. Solarization + 7g/m2 of MBr produces a significant increment in carrots yield when compared with solarization or control with no treatment. The abnormal figure for MBr disinfestation on Tomatoes is not well understood. Crop health monitoring on tomatoes and strawberries did not show special phytopathologycal problems, control treatments on carrots and carnation, were seriously diseased along the crop. Necrotic vessel ratio at the end of the crop for carnations showed good control with fumigation treatments, and intermediate health ratio for solarization. 4. Discussion Solarization with plain mulching does not seem sufficient to control fungi in our geographical area; the efficiency is substantially improved with mulching under tunnel. The improvement is doubtless attributable to higher thermal levels reached (Table 3) as well as to a longer exposure to lethal temperatures. These results show a direct lethal effect on the surviving fungal structures even at the greatest studied depth (24 cm). Absence of significant differences between solarization with fumigants at very low doses and the conventional disinfestation with fumigants at standard doses allow to suggest this combined method as excellent for pathogen control, with the advantage of lower use of toxic products, and consequently, a reduction of residues in the soil; moreover, it shortens safely the minimum exposure time to solarization in a significant way. Tunnel with mulching had, by far, a better performance, than solarization with plain mulching, although its effect in depth is not always sufficient. The method used is valuable for studying the efficacy, of any kind of soil disinfestation treatment against fungi. The results of for adventitious weed control agree with those obtained for fungi, although plain mulching, does not produce a total control, does appear as agronomically valuable for the but next crop, since there is a reduction in competition. The possibility of combining MBr fumigation at reduced dosages with soil solar heating while keeping or even improving the yield is clear upon practical crops. This technique avoids uncertainty to soil solarization on middle latitudes, and would allow to reduce significantly the soil covering time up to 15 days in most instances. 5. Acknowledgments We express our appreciation to Aporta for their kind contribution for MBr dosage in our experimental plots To M. García and E. Serrano, and to Cooperatives of Castelló and l'Alcudia for their kind help with crop experiments. References Busto del A., G. de Barreda D.,Martinez P.F.,Cebolla V., Campos T. 1989. Solarización en la comunidad Valenciana. Sus efectos en malherbología. Proc. 4º EWRS Mediterranean Symposium. V.II. 178-189. Carretero J.L. 1977. Estimación del contenido de semillas de malas hierbas de un suelo agrícola como predicción de su flora adventicia. Anal. Inst. Bot. Cavanilles 34(1):267-278 Cebolla V., M. García. 1984. Desinfección del suelo en cultivo de Fresón al aire libre y bajo invernadero. Publicaciones SEA Moncada (Valencia) Diciembre 1984. Katan J., Greenberger, A., Alon H., Greenstein A. 1976. Solar heating by polyethilene mulching for the control of diseases caused by soil-borne pathogens. Phytopahology 66:683-688 Katan J. 1980. Solar pasteurization of soils for desease control: status and prospects. Plant Disease 64:450-454 Komada H. 1975. Development of a selective medium for quantitative isolation of Fusarium xysporum, from natural soil. Rev. Plant. Prot. Res. 8:114-125 Malathrakis N.E. 1987. EC Joint experts meeting on integrated Pest Management in Protected Vegetable Crops. Cabrils. Spain. 27-29 May. Martinez P.F., Cebolla V., Del Busto A. 1987. El uso de la energía solar para la recuperación de suelos fatigados. Horticultura Profesional 12:85-90 Menzies J.H.,Griebel G.E. 1967. Survival and saprophytic growth of Verticillium dahliae in uncropped soil. Phytopathology 57:703-709 Ponchet J. el al. 1972. Méthodes seléctives d'isolement du Phytophthora nicotianae f.sp. parasitica (dastur) Waterh. a partir du sol. Annales de Phytopathologie 4:97-108 Table 1 - Surviving per cent after 15 days exposure under PE Check 56 28A 14A 14T Mulch Tu F.o.diant. 100f 6 ab 12 abc 5 a 9.8abc 100f 48d R. solani 100f 6.7abc 0.8ab 2 abc 0 a 95f 26d P.parasit. 38cdef 2.4ab 0a 2.4ab 0 a 42cdef 7abc P.cactorum 72def 8 abc 10abc 1 a 3.6ab 45dcdef 27abcd Pythium spp 93f 10 ab 19abcd 6 a 14 abc 74 f 14abcd Table 2 - Surviving per cent after 30 days exposure under PE ----------------------------------------------------------- Check 56 28A 14A 14T Mulch Tu ----------------------------------------------------------- F.o.diant. 100f 10 abc 3 ab 10 a 1.4abc 92f 45d R. solani 100f 14 abc 20abcd 8 a 11 ab 93f 34d P.parasit. 92f 10 abc 0 a 15 abc 0 a 62def 4.8abc P.cactorum 63def 20 abc 1 a 11 ab 2 a 40bcdef 2.8 a Pythium sp. 95f 7 ab 0.5a 6 ab 0.5a 72f 3 ab ----------------------------------------------------------- Table 3 - Exposure time (hours) above a temperature level for each treatment and depth (cm) Mulching Tunnel Check ---------------------- -------------- --------------- Level 4 8 16 32 64 16 32 64 4 8 16 32 ----------------------------------------------------------- >33ºC 848 863 1082 1147 1134 1152 1150 1113 182 112 25 0 >35 644 658 754 784 300 1142 1132 1070 89 61 0 0 >37 502 501 445 128 0 1122 1102 883 55 11 0 0 >39 386 384 168 1 0 1016 996 599 24 0 0 0 >41 289 265 4 0 0 682 571 0 0 0 0 0 >43 173 138 0 0 0 383 47 0 0 0 0 0 >45 43 10 0 0 0 124 0 0 0 0 0 0 >47 0 0 0 0 0 1 0 0 0 0 0 0 >49 0 0 0 0 0 1 0 0 0 0 0 0 ----------------------------------------------------------- Table 4 - Weed control rate evolution ----------------------------------------------------------- Check 56 28A 14A 14T Mulch Tu ----------------------------------------------------------- September 0b 97a 96.3a 98.5a 99.8a 93.5a 97.5a November 0b 95.5a 93a 99.3a 98.7a 93.7a 96.3a May 0d 97.2c 95.8c 93.3c 96 c 69.2a 86.7b ----------------------------------------------------------- Table 5 - Number of seedling spp germinated in greenhouse Check 56 28A 14A 14T Mulch Tu ----------------------------------------------------------- Solanum nigrum 24 0 0 0 0 0 0 Sonchus spp 25 0 0 1 0 3 0 Lolium spp 48 0 0 2 0 1 0 Plantago lanceolata 4 0 0 0 0 0 0 Asphodelus fistulosus 2 0 0 0 0 1 0 Euphorbia exigua 8 0 0 0 0 0 0 Euphorbia prostrata 3 0 0 0 0 0 0 Malva spp 1 0 0 0 0 0 0 Medicago spp 0 0 0 0 0 1 0 Cruciferae spp 0 0 0 0 0 1 0 Non identified 35 5 0 4 3 13 1 ----------------------------------------------------------- Total 150b 5a 0a 7a 3 20a 1a Table 6 - Total yield (Kg/m2) on horticultural crops and on carnation (flowers/m2). ----------------------------------------------------------- Crop Check Mbr(dosage) Solariz + MBr (dosage) Solar --------- ------ ----------- ------------------------ ----- Strawberr 2.8b 3.3a (70g) - 3.5a (17.5g) 3.2b Tomato 4.8bc 4.6c (56g) - 6.6a (14g) 5.8ab Carrot 0.85a - - 7.69c (7g) 4.98b Carnation 5.2a 197b (70g) 202.6b (28g) 206.3b(14g) 177.1b -----------------------------------------------------------