Biological soil disinfestation using ethanol: effect on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis> and soil microorganisms

Better soil disinfestation methods, such as biological soil disinfestation (BSD), that are environmentally safe are increasingly been developed and used because of rising concerns related to environmental risks. We evaluated the efficacy of soil disinfestation using ethanol to control the fungus Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt of tomato. Survival of bud cells and chlamydospores declined markedly in soil saturated with diluted ethanol solution in the laboratory. In field trials, artificially added nonpathogenic Fusarium oxysporum and indigenous F. oxysporum were both strongly suppressed in soil saturated with 1% ethanol solution; a wheat bran treatment was not as effective. The artificially added fungus was not detected in three of four sites treated with ethanol but was detected in three of four sites amended with wheat bran. Using ethanol in pre-autoclaved soil was not suppressive; thus native microorganisms are essential for the suppression. This ethanol-mediated biological soil disinfestation (Et-BSD) temporarily increased the number of anaerobic bacteria, but the number of fungi and aerobic bacteria was stable. Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) analysis revealed slight but apparent differences in bacterial community structures in the soil treated with Et-BSD compared with the structure in soils after other treatments such as water irrigation and in the control soil, which received neither organic amendment nor irrigation after 15 days. Et-BSD is a potentially effective and easy soil disinfestation method, and its impact on native, beneficial microorganisms is moderate.     

Effect of indole-acetic acid (IAA) on the development of symptoms caused by <Emphasis Type="Italic">Pythium ultimum</Emphasis> on tomato plants

The effect of indole-acetic acid (IAA) on the development of symptoms caused by Pythium ultimum on tomato plants was investigated using different bioassays. Application of IAA (5 μg ml⁻¹) on tomato seedlings inoculated with P. ultimum did not affect their emergence suggesting that IAA did not affect the severity of Pythium damping-off. However, IAA was shown to influence the development of P. ultimum symptoms on tomato plantlets. Low concentrations of IAA (0–0.1 μg ml⁻¹) within the rhizosphere of plantlets increased the severity of the symptoms caused by P. ultimum, while higher concentrations (10 μg ml⁻¹), applied either by drenching to the growing medium or by spraying on the shoot, reduced the symptoms caused by this pathogen. In addition, the study demonstrated that P. ultimum produces IAA in liquid culture amended with L-tryptophan, tryptamine or tryptophol (200 μg ml⁻¹) and in unamended culture.     

Nematicidal potential of materials from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal effect of soil amendments with dry top and root material from Medicago sativa and/or Medicago arborea was evaluated on the root-knot nematode Meloidogyne incognita and on the cyst nematode Globodera rostochiensis in potting mixes. All amendments suppressed root and soil population densities of both nematode species compared to non-treated and chemical controls. The suppressiveness of M. sativa differed between top and root material and among the amendment rates. In field conditions soil amendments with 20 or 40 t ha⁻¹ of a pelleted M. sativa meal increased tomato crop yield and reduced soil population densities and root galling by M. incognita. It is suggested that saponins were at least partly responsible for the nematicidal activity.     

Combining sub-lethal heating and on-farm wastes: effects on <Emphasis Type="BoldItalic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="BoldItalic">cumini</Emphasis> causing wilt of cumin

In a 2-year field study, the effects of four heating regimes established by varying temperature or duration of heating on efficiency of on-farm wastes as soil amendments in controlling Fusarium oxysporum f.sp. cumini (Foc) causing wilt on cumin (Cuminum cyminum L.), were ascertained. Significant improvement in reduction of Foc propagules was achieved with the increase in duration and amount of heat. In 2000, mild heating under shade (heat level 4), 31.8–65.9% reduction in Foc propagules was estimated in all the amendments at 0–30 cm soil depth, which improved by 75.7–86.5% in the treatment where Foc-infested soil brought from the laboratory was exposed to direct heat (heat level 3) and amendments and irrigation were applied. Foc propagules were reduced by 76.6–88.3% when infested soil was exposed continuously to dry heat for 56 days (heat level 2), improving efficiency of amendments by 0.9–13.5% compared with heat level 3. After 56 days of exposure to dry heat, elevating the temperature by mulching amended soil with a transparent polyethylene film (50 μm thick) for 20 days (heat level 1) augmented reduction by 80.2–95.5%. In the second season, combining 0.04% of onion, Verbisina encelioides or mustard oil-cake with mustard residues (0.18%) improved the reduction in Foc propagules at all heat levels even at the lower soil depth. Maximum reduction (94.9–100%) in Foc propagules at heat levels 1 to 3 was achieved when residues of Verbisina were supplemented with those of mustard. These results suggest a new approach to improve the control of Foc by combining prolonged heating with on-farm wastes such as Verbisina residues and one summer irrigation.     

Application of sugar beet vinasse followed by solarization reduces the incidence of Meloidogyne incognita in pepper crops while improving soil quality

The application of organic amendments followed by solarization is a promising alternative to chemical disinfestation for the control of soilborne pathogens in protected crops. We assessed the effectiveness of the application of sugar beet vinasse, alone and in combination with fresh sheep manure, followed by solarization on (i) disease incidence of Meloidogyne incognita in protected pepper crops of southeast Spain and (ii) soil properties with potential as indicators of soil quality. When treatments were applied in summer, a 97% and 87% reduction in the galling index and percentage of galled plants, respectively, was observed in vinasse-amendment plots at the end of the crop season, compared with untreated controls. Treatments led to higher values of soil organic C, total N, total P and extractable K⁺, and lower values of soil pH. Values of enzyme activities (dehydrogenase, β-glucosidase, acid and alkaline phosphatase, arylsulfatase, urease), microbial biomass C, potentially mineralizable N, water soluble organic C and microbial functional diversity were higher in treated versus untreated soils. Positive correlations were found between enzyme activities and microbial biomass C. In general, no differences were observed between vinasse and vinasse+manure treatments. It was concluded that the application of sugar beet vinasse followed by solarization is a good alternative to methyl bromide for the control of M. incognita in protected pepper crops of southeast Spain, especially, but not exclusively, when carried out in summer. Additionally, treatments resulted in an improvement in soil quality, as reflected by the higher values of microbial biomass, activity and functional diversity.     

Effect of variety choice, resistant rootstocks and chitin soil amendments on soil-borne diseases in soil-based, protected tomato production systems

Soil-borne diseases are the most significant crop protection problem in soil-based, low-input and especially organic glasshouse production systems in Europe. While chemical soil disinfestation has been the control method of choice in conventional farming systems, soil steaming has been the main strategy for the control of soil-borne diseases in organic production. Both methods are extremely expensive and have been increasingly restricted for environmental reasons by governments, and integrated and organic farming standard-setting bodies. The use of disease-tolerant varieties, grafting onto resistant rootstocks and chitin soil amendments were evaluated as potential replacements for soil steaming in organic and other low-input tomato production systems. When only Pyrenochaeta lycopersici and/or Meloidogyne spp. were present in soil, grafting and/or chitin soil amendment were found to be as effective in reducing root disease and/or increasing yield as soil steaming, but the efficacy of both treatments was reduced when Verticillum albo-atrum was also present in soil. No additive effects of combining grafting and chitin soil amendments could be detected. A more widespread use of grafting and/or chitin soil amendments may therefore allow significant reductions in the use of steam and chemical soil disinfestation in glasshouse crops. It will also allow integrated and organic farming standard-setting bodies to impose further restrictions on the use of soil disinfestation treatments.     

Effect of solarization and fumigant applications on soilborne pathogens and root-knot nematodes in greenhouse-grown tomato in Turkey

A study was conducted in two greenhouses with a history of Fusarium crown and root rot (Fusarium oxysporum f.sp.radicis-lycopersici, Forl) and root-knot nematodes (Meloidogyne javanica andM. incognita). During the 2005–06 growing season, the effectiveness of soil disinfestation by solarization in combination with low doses of metham-sodium (500, 750, 1000 and 1250l ha⁻¹) or dazomet (400 g ha⁻¹), was tested against soilborne pathogens and nematodes in an attempt to find a suitable alternative to methyl bromide, which is soon to be phased out. Solarization alone was not effective in the greenhouse with a high incidence ofForl. In the greenhouse with a low level ofForl, all the treatments tested reduced disease incidence, and were therefore considered to be applicable for soil disinfestation. In addition, root-knot nematode density decreased with all the treatments tested in both of the greenhouses.     

The potential for Fusarium oxysporum f. sp. fragariae,cause of fusarium wilt of strawberry,to colonize organic matter in soil and persist through anaerobic soil disinfestation

Fusarium wilt of strawberry, caused by Fusarium oxysporum f. sp. fragariae, is a disease of primary concern for strawberry production in many countries. Crop rotation and anaerobic soil disinfestation (ASD) have gained recent interest for their potential to contribute to management of this disease. Both techniques involve incorporation of organic matter into soil, which may be utilized by strains of Fusarium that are competitive saprophytes. We show that F. oxysporum f. sp. fragariae can colonize strawberry, lettuce, raspberry, and broccoli leaf tissues, which are sources of organic matter generated during crop rotation. This pathogen increased in soil population density during ASD treatments that did not become anaerobic, possibly as a result of growth on the organic amendment. However, significant population decreases were observed after ASD treatment when at least 100,000 cumulative reduced mV hours occurred in a 14-day experiment. Post-ASD abundance of F. oxysporum f. sp. fragariae in soil was negatively correlated with cumulative reduced mV hours. The only treatment that consistently caused disinfestation was exposed to a maximum temperature of 22 °C, which indicates there is potential for developing effective ASD treatments in the cool climates where strawberries are grown. Awareness that F. oxysporum f. sp. fragariae can act as a competitive soil saprophyte should be further investigated for its potential to alter disease outcomes where organic amendments are applied.     

Reduced sclerotial viability of Stromatinia cepivora and control of white rot disease of onion and garlic by means of soil bio-solarization

The suppressive effects of soil bio-solarization, which is a new method of soil disinfestation that combines soil bio-fumigation with soil solarization, against the sclerotial viability of Stromatinia cepivora and the subsequent control of white rot disease of onion and garlic were evaluated. Soil was bio-fumigated with fresh amendments of cow manure, chicken manure, horse manure, cruciferous plant residues, or Allium waste, at 30,000 kg/ha. After bio-fumigation, the soil was irrigated and covered with a 200 μm transparent plastic sheet for 60 days. Plots that received fresh amendment and remained uncovered and untreated served as controls. Solarization alone increased the maximum soil temperature to 55.3 °C, 50.3 °C and 46.3 °C at 10, 20, and 30 cm depths, respectively, which led to significant reductions (98.0%, 89.3%, and 62.7%, respectively) in the sclerotial viability of S. cepivora. Soil bio-solarization with cruciferous plant residues or Allium waste resulted in the strongest negative effects on the sclerotial viability of S. cepivora, with reductions of 100.0%, 98.7%, and 87.3% or 100.0%, 99.3%, and 87.7%, at 10, 20, and 30 cm depths, respectively. Compared to the non-treated control, these treatments significantly reduced the incidence of white rot disease in onion and garlic, which led to increases in onion and garlic yield in fields that were heavily infested by S. cepivora.

     

Use of different plastics for soil solarization in strawberry growth and time–temperature relationships for the control of <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> and weeds

The aim of this study was to determine the effect of soil solarization on soilborne diseases, weeds and plant yields by using polyethylene film (30-μm-thick) containing different additives [ultraviolet (UV), ultraviolet + infrared (UV + IR), ultraviolet + infrared + anti-fog + anti-dust (UV + IR + AF + AD)], and used polyethylene film (260-μm-thick). Trials were conducted in commercial strawberry (Fragaria ananassa cv. ‘Camarosa’) fields in the town of Sultanhisar in Aydin province, Turkey, between 2007 and 2009. The highest soil temperatures at the depth of 10 cm under a polyethylene sheet containing UV + IR + AF + AD were 54°C in 2007 and 50.7°C in 2008. During the 2007 growing season, collapse and death of strawberry plants were not detected. At the end of the 2008 season (May–June), collapsed and dying strawberry plants were observed. Pure cultures of Macrophomina phaseolina and Rhizoctonia solani were isolated from affected roots and crowns of plants. Viability studies of M. phaseolina were conducted under various field conditions and temperatures and M. phaseolina sclerotia survived more than 18 days at 45°C. There was a sharp decline in M. phaseolina at 50°C, where it survived for 19 h but was completely killed at 20 h. It first lost viability after 17 h at 50°C and after 60 min at 55°C. In the field, solarization did not reduce the viability of M. phaseolina at a soil depth of 10 or 20 cm; however, a significant reduction (66%) in survival was determined at a soil depth of 5 cm. All treatments controlled Portulaca oleracea, Amaranthus spp., Digitaria sanguinalis, Echinochloa crus-galli, Veronica hederifolia, Raphanus raphanistrum, Setaria verticillata and Mercurialis annua at a rate of 100%, but no treatment was effective on Cyperus rotundus. The marketable fruit yield was 38,004 kg.ha⁻¹ for UV + IR, 35,834 kg.ha⁻¹ for UV-added polyethylene film and 35,368 kg.ha⁻¹ for used polyethylene sheet-covered plots, whereas it was 27,365 kg.ha⁻¹ for untreated control plots.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits the plant cell wall degrading enzymes of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> and reduces the severity of groundnut stem rot

Three hundred and ninety-three groundnut-associated bacterial strains, applied both as seed treatment and soil amendment, were evaluated for control of stem rot disease (caused by Sclerotium rolfsii) of groundnut in a controlled environment. Twelve strains significantly (P=0.01) reduced the incidence of stem, rot of which groundnut seed endophytes Pseudomonas aeruginosa GSE 18 and GSE 19 reduced the seedling mortality by 54% and 58%, compared to the control. In dual cultures, the 12 biocontrol strains reduced the mycelial growth of S. rolfsii by 32%–74% as compared to the control. Cell- free culture filtrates of P. aeruginosa GSE 18 and GSE 19 inhibited the activity in vitro of the cell wall-degrading enzymes (CWDE) polygalacturonase and cellulase by S. rolfsii up to a maximum of 55% and 50%, respectively, when measured 6 days after inoculation. Pseudomonas aeruginosa GSE 18 and GSE 19 with a known tolerance to thiram, a commonly used seed dressing fungicide, suppressed the growth of S. rolfsii, inhibited the activity of CWDE, and reduced the incidence of stem rot, suggesting the usefulness of these biocontrol strains as components in the integrated management of groundnut stem rot.     

Introduction of a new agricultural technology —Soil Solarization—in Israel

Soil solarization is a non-chemical method for soil disinfestation. Theoretically, Israel is one of the ideal sites for the use of this technology. It has been shown that the introduction of soil solarization is highly crop-dependent and is inversely correlated with the availability of alternative soil disinfestation methods. Solarization has been easily introduced for crops with no reliable and recommended soil disinfestation methods or in organic farming and in cases where the technique is inexpensive. Its introduction into crops for which other soil disinfestation techniques are established, or to less intensive crops, has been much slower. Apart from objective reasons for not using solarization,e.g. plots which are in use during the suitable season or low efficacy against specific soilborne pests, there are problems that need to be solved. These are related to technology and technology diffusion, to the price of the plastic sheeting, and to the priority given by farmers to non-chemical methods.     

Site-specific field resistance of grapevine to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> correlates to altered gene expression and was not modulated by the application of organic amendments

The influence of site on resistance of grapevine (cv. Chasselas) to Plasmopara viticola was evaluated. Grapevine leaves from three vineyards in the region of Lake Neuchatel (Switzerland) were tested for their susceptibility to P. viticola in the lab in five successive years (2004–2008), and the expression levels of four selected defence-related genes (Glucanase, Lipoxygenase 9, 9-cis epoxycarotenoid dioxygenase, Stilbene synthase) were studied in 1 year. In all 5 years of examination, differences between sites were substantial. In four out of 5 years, plants from site Hauvernier were much less susceptible to P. viticola than plants from site Auvernier. In another year, differences were less pronounced but still significant for one leaf age. Susceptibility of plants from a third site (Concise) varied from year to year. Differences in the genetic background were excluded by microsatellite analysis. Differences in susceptibility were mirrored in the constitutive expression pattern of four defence-related genes, with samples from the Hauterive site clearly separated from samples of the other two sites in redundancy analysis. Furthermore, it was evaluated whether site-specific resistance can be modulated by agronomic practices such as the application of organic amendments. In two commercial vineyards (cv. Pinot noir), soils had either not (control) or yearly (compost) been amended with a compost for the last 9 years. Leaves from plants grown in any of the two treatments did not differ in their susceptibility to P. viticola in both years of examination. Additionally, under controlled conditions, none of 19 different composts amended to the substrate of grapevine seedlings or cuttings affected their susceptibility to P. viticola, but 8 out of 19 composts reduced severity in the control bioassay Arabidopsis thaliana—Hyaloperonospora arabidopsidis, indicating that a modulation of site-specific susceptibility of grapevine plants by organic amendments is at the very least, difficult.     

Biofumigation with Brassica plants and its effect on the inoculum potential of Fusarium yellows of Brassica crops

The use of Brassica crops as green manure in the so-called biofumigation treatment has been successfully exploited for the management of soilborne pathogens and is gaining interest particularly in the case of less intensive agricultural systems. A study was undertaken to investigate possible negative side-effects of biofumigation in order to prevent possible damage caused by wilt pathogens able to attack both plants used for biofumigation as well as agricultural crops. To do so, firstly the response of different Brassicas, including some used in biofumigation, to the formae speciales of Fusarium oxysporum known for being pathogenic on Brassica crops was evaluated. Secondly, the effect of green manure treatments on yield, quality of crops, and inoculum densities, infection and survival of Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani was evaluated. In the second part of the work, four Brassica crops, selected for their response (susceptibility or resistance) to F. oxysporum f. sp. conglutinans and to F. oxysporum f. sp. raphani were evaluated in order to determine their response to the two pathogens during subsequent crops grown in soil where plants were incorporated as green manure into the soil at the end of each cycle. Moreover, the dynamics of the populations of F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani in the soil after several biofumigation cycles was studied. Many of the Brassica crops used for biofumigation tested were susceptible to F. oxysporum f. sp. conglutinans and or to F. oxysporum f. sp. raphani. Green manure treatment, carried out by growing nine cycles of biocidal plants, with a short crop cycle of 30–35 days, did not reduce Fusarium wilts on susceptible Brassica hosts. The population of the pathogen was partially increased as a result of the incorporation of tissues of the susceptible plants. When Brassica crops grown were resistant to the two F. oxysporum pathogens used for soil infestation, green manure simulation did inhibit both pathogens, thus confirming its biocidal activity. The results obtained under our experimental conditions show that biofumigation treatment is not applicable for soil disinfestation on crops susceptible to the same formae speciales of F. oxysporum affecting Brassica species used for biofumigation. Brassica crops resistant to Fusarium yellows should be grown where biofumigation is applied. Moreover, alternation of crops used for biofumigation should be encouraged.     

Soil solarization as a substitute for methyl bromide fumigation in greenhouse tomato production in Cyprus

Preplant soil fumigation with methyl bromide (MB) is presently standard practice in greenhouse tomato production. Since this compound is scheduled to be phased out by 2005, the possibility of using solarization as an alternative soil disinfestation method was examined in four greenhouse tomato trials. Solarization was applied for 8 weeks in July-August, using transparent polyethylene sheets for soil mulching, and compared with MB fumigation applied in September, before planting, at 80 g/m². Solarization raised the maximum soil temperature by 9°C and reduced the population density ofFusarium spp. in soil by 91–98%. Similar reductions of soil inoculum (95–99%) were obtained with MB fumigation. Both methods provided effective control of Fusarium wilt, Verticillium wilt and corky root rot on tomato plants. MB fumigation was in addition highly effective against root-knot nematodes, whereas nematode control with solarization did not exceed 50%. Both treatments resulted in similar fruit yield increases, ranging within 90–140% compared with plants grown in untreated soil. During the second cropping season following soil treatment, solarization exhibited two times higher residual effectiveness against vascular wilt diseases compared with MB fumigation. The latter treatment, however, was superior to solarization in its residual effectiveness against root-knot nematodes and to a lesser extent against corky root rot. Fruit yields from solarized and MB-fumigated soil during the second cropping season were higher than those obtained from untreated soil by approximately 35% and 60%, respectively. In Cyprus, solarization appears to be an effective alternative to MB fumigation in greenhouse tomato production, especially if integrated with other approaches enabling more effective nematode control.     

Effect of seed treatment of<Emphasis Type="Italic">Arachis hypogaea</Emphasis> with<Emphasis Type="Italic">Bacillus subtilis</Emphasis> on nodulation in biocontrol of southern blight (<Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>) disease

The employment of formulatedBacillus subtilis for peanut seeds (Arachis hypogaea cv. ‘Shulamit’) counteracted the destructive effects of the seedborne pathogenSclerotium (Athelia)rolfsii on the nodulation, leghemoglobin and nitrogenase activity of peanuts. Moreover, the changes in crop vigor index, total nitrogen content and survivability of bothRhizobium spp. andB. subtilis have been related to compatibility and even an occasional synergism between them. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Evaluation of methyl iodide as a soil fumigant in container and small field plot studies

Methyl iodide was evaluated as a soil fumigant as a potential replacement for the widely used soil fumigant methyl bromide. In container trials, methyl iodide was significantly more effective than methyl bromide against the plant parasitic nematodes Meloidogyne incognita, Heterodera schachtii and Tylenchulus semipenetrans and the plant pathogenic fungus Rhizoctonia solani. In small field plots, soil populations of root-knot nematodes were no longer detected after methyl iodide fumigation at an application rate of 112 kg ha^-1. However, after growing a susceptible lima bean host for two months, substantial root-knot galling occurred, while Rhizobium nodulation was absent. At 168 kg ha^-1 of methyl iodide, root-knot galling was reduced to less than 1%, and no Pythium propagules were recovered on selective detection media. These efficacy data support the conclusion that methyl iodide is a likely candidate for replacing methyl bromide as a soil fumigant. © 1998 SCI.     

Zur Kenntnis der Blattminen-MotteCameraria ohridella Desch. & Dim. (Lep., Lithocolletidae) anAesculus hippocastanum L. in der Tschenchischen Republik

Cameraria ohridella was recorded first in 1985 in Macedonia. It gradually expanded to north and west and at present it is a serious pest of Horse ChestnutAesculus hippocastanum in the Czech Republic, having been established at about 80 localities. There are 4–5 overlapping generations with sizes of the larvae of 0.4–4.0 mm. The larva develops inside the leaf tissue in the upper parenchym layer of the leaflet and causes a mine, the size of which is broadened with growing larva. First adults start to fly at the end of April. After mating the females lay single eggs on the leaves. Larval development lasts 25–30 days followed by the prepupa I and II. The latter spins a cocoon in which the pupa of the last generation hibernates. The development from L4 to the prepupa lasts for 3–6 days at 22°C. During the summer it is possible to find all larval and prepupal stages in attacked leaves. FourAesculus species:A. parviflora Walk,A. carnea Haey.,A. glabra Walk andA. indica J. Hobb. were found to be resistent toC. obridella. A. lutea H. J. was liftle andA. pavia L. was heavily attacked. The parasitization ofC. o. larvae was very low. Only 2 parasites were found in 1500 mines in the first and second generation ofC. o., and 40 parasites in 1000 mines of the fourth and fifth generation. The highest mortality takes place in moths, eggs and young larvae. It was found in all 4 generation that there were i. m. 50 eggs/leaflet from which i. m. 3 hibernating pupae resulted. Supposing 2 moths (1♀, 1♂)/leaflet emerged in spring which produced 50 eggs (75 eggs/♀—33% moth mortality), the density of eggs in the first generation after hibernating being the same as in the last (fourth) generation before hibernating. As to the whole populations density in this case we can calculate as following: 3 pupae/leaflet on the tree=3000 pupae/leaflets pro m² on the soil=2000 emerging moths pro m²=50,000 moths (25,000 ♀♀) pro tree (namely 25 m² projecting area of the tree crown×2000 moths).