Petr Fojtik¹, Fredrik Schlyter², Jan Vrkoc³, Pavlina Vrkocova³

The large pine weevil, Hylobius abietis L., is known to be a major pest of reforestation in Europe. The adult insects feed on the bark of young trees, particularly on conifers, causing the death of the plants when they are girdled. Serious damage may occur for up to three years after planting out of seedlings in the field. If plants are not protected, an average of 40 to 50% loss may be expected. Current control methods rely heavily on pyrethroid insecticides. Although research on some new slow release natural repellent formulations suggest scope for improved control, this can only be achieved at relatively high cost. In laboratory and field tests, carvone formulations exhibited best and promising results. Several approaches to control are possible and this project will be a platform for the introduction of non-host repellents into integrated pest management program.

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¹ Forestry and Game Management Institute, Jiloviste-Strnady, Czech Republic

² Department of Plant Protection Sciences, Swedish Agricultural University, S-230 53 Alnarp, Sweden, <Fredrik.Schlyter@vsv.slu.se>

³ Department of Natural Products, IOCB ASCR Flemingovo nam. 2, CZ-166 10, Praha, Czech Republic, <vrkocova@uochb.cas.cz>

Fernando Petacci¹, João Batista Fernandes¹, Edson Rodrigues Filho¹, Paulo Cezar Vieira¹ and Maria Fatima G. F. da Silva^1, Odair Corrêa Bueno², Maria José Hebling², Fernando Carlos Pagnocca², Maurício Bacci Jr²

Some ants are known as leaf-cutting ants and Atta and Acromyrmex are considered the most evolved genera between them. These ants have a fungus associated with them which is the main food source for broods. The fungus use the vegetal material for growing by a mechanism of polysaccharides degradation.

In the Atta nests are observed accumulation of old fungus mass together with died ants and other material refused, which are commonly named as garbage.

This work is a preliminary evaluation of micro-nutrients occurring in the organic material found inside the nest, using classical methods of chemical analysis.

The Table 1 shows the quantitative differences between metals, phosphorus, carbon, Sulfur, hydrogen, nitrogen and ash concentration in the samples analyzed.

Table 1: Concentration of metals (ppm), phosphorus, carbon, sulfur, hydrogen, nitrogen and ash (%) in the fungus garden and garbage.

Chemical Elements	fungus garden	ant garbage
Fe Zn Mg Na Ca B K Mn P N C H S Ash	140 82 4894 1124 6430 170 15030 768 0.833 5.34 47.02 6.34 0.76 5.37	259 156 7069 1870 14650 163 15260 1366 0.743 4.67 49.23 6.29 0.67 7.46

The difference of 11.8, 10.8 and 12.5 % between percentages of sulfur, phosphorus and nitrogen, respectively, indicate that ant garbage is poorest in these elements than nest material and suggest that they are consumed in the nest. It is observed the enhancement of near 50% of metals amounts in the ant garbage, with exception of potassium and boron.

FAPESP/CNPq/CAPES/PRONEX-MCT-FINEP

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¹Departamento de Química - UFSCar _ rodovia Washington Luiz, Km 235, C. P. 676, São Carlos _ SP

²CEIS _ UNESP, av. 24 A, 1515, Rio Claro _ SP

Roberto T. Saito¹, Rubens C. L. Figueira², Moyses G. Tessler³, Ieda I. L. Cunha¹

²¹⁰Pb and ²¹⁰Po are natural radionuclides present in the environment in trace quantities. From the seawater, they can be incorporated by the bottom sediment or be assimilated by the aquatic organisms. In this case, there is a transfer through a food chain, with a tendency of higher accumulation in the upper levels. Consumption of aquatic organisms is one of the main pathways by which radionuclides can enter to the human organism. In this work, ²¹⁰Pb and ²¹⁰Po analyses were carried out in seawater, sediments, fish, shellfish and crustaceans collected in the estuary. For ²¹⁰Pb analysis via ²¹⁰Bi, samples were dissolved by acid lixiviation (or digestion), lead was purified by successive steps of precipitation and dissolution. Lead sulphate was counted by employing a Geiger-Müller detector. ²¹⁰Po analysis consisted of several steps: acid digestion, polonium deposition onto a silver disk and detection by alpha spectrometry (silicon detector). ²¹⁰Pb and ²¹⁰Po levels in superficial water samples ranged from 5.5 to 10.8 mBq.L^-1 and from 4.7 to 8.6 mBq.L^-1, respectively; in fish muscle ranged from minimum detectable concentration (M.D.C. = 0.58 Bq.kg^-1) to 1.84 Bq.kg^-1 and from 0.15 to 8.76 Bq.kg^-1, respectively. In crustaceans, the levels found varied from 2.9 to 3.8 Bq.kg^-1 for ²¹⁰Pb and from 10.4 to 33.4 Bq.kg^-1 for ²¹⁰Po. For shellfish, levels of ²¹⁰Pb and ²¹⁰Po ranged from 4.5 to 7.4 Bq.kg^-1 and from 52.5 to 148.6 Bq.kg^-1, respectively. These values are in good agreement with the values published at the literature and represent reference values to the brazilian coast, forming a data base. By employing statistic analysis on the radionuclide levels obtained for fish, it was not noticed correlation between the radioactivity levels and fish ecological niche . This can be consequence of the small depth of the estuarine channels (about 7 m) as well as of both the resurgence constant and mixture of thinner sediments due to the tidy currents and of the influx of water from the local rivers. Probably, in the shallow estuarine regions, as the Cananeia-Iguape estuary, ²¹⁰Pb and ²¹⁰Po accumulate at random in fish, however, filtering organisms, such as shellfish, the feeding habits has great influence in the radionuclide accumulation, showing that the sediment is an important contributor to the assimilation of natural radionuclides by the human beings.

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1- IPEN/CNEN-SP _ Supervisão de Radioquímica; 2 - CETEC _ Universidade Cruzeiro do Sul; 3 - Instituto Oceanográfico, Universidade de São Paulo

05422-970 - Cid. Universitária - São Paulo _ Brasil

E-mail: biologia@net.ipen.br

TAE-YOUNG KIM·BONG-SEOP KIL*·BYOUNG-IN DOO·KEW-CHEOL SHIM** AND JI-HYUN KIL***

Seed germination of the receptor species were inhibited by the aqueous extracts of donor plant and these inhibitory effects were increased in proportional to the concentration of extracts. But, seedling elongation of the receptor species represented different results. One of the receptor species were inhibited by the aqueous extract increased, another species were slightly stimulated in lower concentration of the extract, and the others were stimulated by the extract increased. Dry weight growth was also proportionally inhibited by the aqueous extract increased, while some species was stimulated in lower concentration of the extract. Seed germination and dry weight growth of the receptor species were not inhibited by the volatile substances of donor plant. But, seedling elongation of some receptor species were inhibited by the volatile substance of donor plant. Effect of C. boreale on roots hair development of receptor species was tested and the result was inhibited along to the concentration of aqueous extracts and volatile substances. Anti-fungal effect of C. boreale on Aspergillus nidulans, Fusarium oxysporum, Alternaria mali was inhibitory along to the concentration of volatile substances increased. Anti-bacterial effect of C. boreale on Staphylococcus aureus, Streptococcus pyogenes and Streptococcus mutans tested for growth inhibition as increased to the concentration of volatile substances. While, growth of Escherichia coli was not inhibited to all the concentration of volatile substances. The above mentioned results were, therefore, confirmed that the natural substances from C. boreale had biologically toxic activities, that is, allelopathic effects to the other plants and microorganisms.

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DEPPARTMENT OF NEWLY-DEVELOPED DRUGS, PROFESSIONAL GRADUATE SCHOOL OF ORIENTAL MEDICINE, *DIVISION OF LIFE SCIENCE, WONKWANG UNIVERSITY, **INSTITUTE OF SCIENCE EDUCATION KONJU NATIONAL UNIVERSITY, ***ENVIRONMENTAL ECOLOGY DIVISION, NATIONAL INSTITUTE OF ENVIRONMENTAL RESEARCH, REPUBLIC OF KOREA

Alberto Luiz Marsaro Júnior¹, Terezinha Maria Castro Della Lucia¹, João Batista Fernandes² and Rejane Carla de Souza²

It is well known that leaf-cutting ants are severe pests of forests and agricultural crops in Brazil. Previous works has shown that the leaf-cutter Atta sexdens rubropilosa exihibted an aggressive behavior among nestmate workers, whenever they were exposed to leaves of Eucalyptus maculata in the foraging arena. This study proposed to further investigate this phenomenon. Chemical samples of epicuticular waxes, essential oils, hexane, dichloromethane, ethyl acetate and methanol extracts were obtained from the leaves and tested on five laboratory colonies of this ant species. Epicuticular waxes did not alter the ants behavior but all other samples showed some activity, hexane extracts being the most active. After a series of fractionings, the sesquiterpenes Elemol and b-eudesmol were identified. Using this last compound the entire behavioral sequence of the ants was described. We found that these sesquiterpenes impregnated the ants' antennae and so contaminated them that they interfered with their capacity of recognizing nestmates, leading to aggressive behavior. Within the interval of 0.0 to 16.67 mg/cm² of b-eudesmol on filter paper, increases in concentration promoted increases in both number of aggression groups and percentage of mutilated ants.

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¹ Universidade Federal de Viçosa, Departamento de Biologia Animal, 36571.000 Viçosa, MG, Brasil

² Universidade Federal de São Carlos, Departamento de Química, São Carlos, SP, Brazil

Yoko Takematsu¹, Nao Fujiwara¹, Ryohei Yamaoka¹, Fujio Hyodo², Nit Kirtibutr³ & Takuya Abe²

Termite nests are classified into 4 types; arboreal nests, epigeal nests, hypogeal nests, and nests in wood. It is generally believed that each species of termite has its own specific type of nest. However, we found that there are all the four types of nests in dry evergreen forest (DEF) of Thailand and that those nests were made by a single genus Microcerotermes. According to conventional belief, this implies that Microcerotermes may have more than four species in the forest. Indeed, five species have been recorded from Thailand, but little is known about the relationship between species and nest types.

Then in this study, we analyzed the cuticular hydrocarbons of Microcerotermes to identify the species and clarified the species composition in conjunction with nest types. The study of nest distribution and nest types was carried out at a dry evergreen forest in Sakaerat Environment Research Station of Thailand. Species of each nest type were identified by analyzing the cuticular hydrocarbons in addition to the conventional morphological observation. Cuticular hydrocarbon analysis was performed on GC and GC/MS.

As an unanticipated result, we found that all colonies collected from four nest types showed the same cuticular hydrocarbon components and they would be the same species. This indicates that one species can have different types of nests. The results of nest distribution indicated that nests of Microcerotermes were most abundant in this forest. Then the reasons for such nest diversity are discussed.
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1 Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan

2 Center for Ecological Research, Kyoto University, Otsu, 520-2113, Japan

3 Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand.

Alexsandro A. Silva¹, Gulab N. Jham¹, Eraldo R. Lima², Roberson L. Necchi² , Paulo A. Viana³, Evaldo F. Vilela²

In the last decade several essays were made in Brazil in order to monitor populations of Elasmopalpus lignosellus with pheromone traps. The negative results of these experiments indicate that Brazilian population may use a different blend or additional compounds for the sexual communication. This reinvestigation of the sex pheromone of E. lignosellus was carried out to determine the optimal blend for male captures for monitoring purposes. Extractions of pheromone glands in hexane were made from 30 virgin females, during the calling period. A GC-EAG, EAG and GC-MS system were used to identify the active substances. The compounds Z9-tetradecenyl acetate and Z11-hexadecenyl acetate were found in all samples of glands. These compounds were active in the antenna of males. In the screening tests these acetates produced the best EAG responses. Based on these results a series of wind tunnel tests are underway to determine the best blend to be used in field conditions.

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¹Dept^o de Química/CCE/UFV C36571-000, Viçosa, MG. ²Dept^o de Biologia Animal/CCB/UFV CEP 36571-000, Viçosa, MG. ³Embrapa Milho e Sorgo, C. Postal 151, CEP 35701-970, Sete Lagôas, MG. E-mail: gulab@mail.ufv.br aaraujos@alunos.ufv.br

*Hiromi. Sasagawa^l, F.-L. Hu ², Y.-J. Hua³ K. S. Boo⁴, and Shigeru Matsuyama⁵.

The Japanese honeybee (Apis cerana japonica Rad) and the introduced European honeybee (Apis mellifera L.) share the same habitat in Japan. Very little is known about the biology of A. c. japonica. The red and white flower varieties of the oriental orchid, "Kinn-ryou henn" (Cymbidium floribundum Lindl.: Cf), has been observed to attract workers, drones, queens even the entire swarming colony of A. c. japonica. However, the orchid does not attract A.. mellifera. This is due to the fact that the flower scent mimics the Nasonov and mandibular glands pheromones of A. c. japonica. Both Nasonov gland and mandibular glands extracts induced aggregation behavior in both bee species, but the GC profiles of the extract from A. mellifera and A. c. japonica were significantly different. It is interesting that a plant like C. floribundum can affect the social behavior of A. c. japonica. The experiments using other Asian honeybee such as Apis cerana cerana are underway.

We report here for the first time that: (1) The same species of C. flooribundum is found in China in 1999. (2) In search of keys to socialities and diversity in insects, the components of pheromone glands (Nasonov, mandibular, and the sting complex) of honey bees in Asia (including 5 species. and 4 subspecies: A. cerana japonica, A. c. cerana, A. c. indica, A. c. himalaya, A. andreniformis, A. laboriosa, A. dorsata, Apis mellifera) were analyzed by GC/MS. In this paper, we summarize our recent findings on the chemistry of pheromone glands of the Asian honey bees.

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¹National Institute of Sericultural and Entomological Science, 1-2 Oowashi, Tsukuba. Ibaraki 305-8634, Japan.(Tokyo Metropolitan Inst. for Neuroscience, PRESTO JST), E-mail sasagawa@nises.affrc.go.jp; ²College of Animal Sciences, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China, Zhejiang University, Kaixuan Road 268, Hangzhou 310029,China, ³Institute of Nuclear-Agricultural Sciences, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China, ⁴Dept. of Agricultural Biology, College of Agriculture & Life Sciences, Seoul National University Suwon 441-744, REP. Korea, and ⁵Inst. of Applied Biochem., Univ. of Tsukuba. Tsukuba. Ibaraki 305-8572, Japan.

Patricia T. Baraldi, Paulo H. G. Zarbin, Paulo C. Vieira, Arlene G. Corrêa

The use of pheromones in integrated pest management has been increasing in the last years due to environmental concern. This development is accompanied by the search for simple, efficient and less aggressive synthetic methodologies for the preparation of pheromones.

One of these methodologies includes microbiological reactions, more specifically, biocatalytic reduction of carbonyl compounds using baker's yeast (Saccharomyces cerevisiae, BY ). This is one of the most inexpensive methods to prepare enantiomerically enriched alcohols, .

2-Methyl-4-octanol (1) has been published as a possible aggregation pheromone component of sugarcane borers, Metamasius hemipterus¹ and Sphenophorus levis.² The synthesis of both enantiomers of 1 is necessary to establish the absolute configuration of the naturally occuring product and also to clarify the relationship between stereochemistry and pheromone activity. Mori et al. have published the synthesis of the enantiomers of 1 from (R)- and (S)- leucine in 5 steps.³

In this work, we present the first enantioselective synthesis of (R)- and (S)-2-methyl-4-octanol from commercial isobutyric acid in 6 steps. The key step of our synthetic route is the asymmetric reduction of b-ketoester 4 with S. cerevisiae affording (S)-5 in high yield and enantiomeric excess. This alcohol was then efficiently converted to (R)-1(Scheme 1).

Scheme 1:

(S)-2-Methyl-4-octanol (1) was prepared from (R)-5, which was obtained from (S)-5 employing a Mitsunobu reaction (Scheme 2).

Scheme 2:

1.Perez, A. L., et al., J. Chem. Ecol. 23: 869, 1997.

2.Zarbin, P.H.G, et. al., unpublished results.

3. Takenaka, M.; Takikawa, H.; Mori, K., Liebigs Ann., 1963, 1996.

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Department of Chemistry, ¹ Universidade Federal Federal de São Carlos , CP 676

13565-905 - São Carlos, SP - Brazil

Jardel A. Moreira, Maurício L. Vieira and Arlene G. Corrêa

The coffee leaf miner, Perileucoptera coffeella (Lepidoptera, Lyonetiidae) is the most serious pest on coffee in Brazil. In 1988, Francke isolated and identified 5,9-dimethylpentadecane (1 ) as the major sex pheromone component of P. coffeella,¹ although the stereochemistry remains to be determined. Few synthesis of this compound as diastereoisomeric mixtures have been published. Thus, the synthesis of all of the four possible stereoisomers of this alkane is necessary to establish the absolute configuration of the naturally occurring pheromone and also to clarify the relationship between absolute configuration and the bioactivity of the chiral pheromone.

In this work, we describe the enantioselective synthesis of (5S,9S)-5,9-dimethylpentadecane from commercial available (-)-isopulegol (2) in 12 steps (Scheme 1).² The stereogenic center at C-5 was established through a stereoselective hydroboration of 2, with a diastereoisomeric ratio of 7:1. The two stereoisomers of 3 formed were readily separated by flash column chromatography.

Scheme 1:

Using the same methodology described above, we are preparing the (5S,9R)- and (5R,9S)-1 from (+)-neo-isopulegol, which can be obtained from (-)-isopulegol in 2 steps. The last isomer, (5R,9R)-1, would be prepared from commercial available (+)-isopulegol.

¹Francke, W., et al. Z. Naturforsch. 1988 , 43C, 787.

² Ferreira, J.T.B.; Zarbin, P.H.G., Bioorg. Med. Chem. 1996, 4, 381.

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Departamento de Química, Universidade Federal de São Carlos, CP 676

13565-905 - São Carlos _ SP, Brazil

Eraldo R. Lima, Vivian D. Motta, Aline S. Bernardino, Roberson L. Necchi, Ana Paula Silva and Evaldo F. Vilela

The coffee leafminer Leucoptera coffeella is a major pest of coffee plantations in Brazil. A large project to study the pheromone ecology is underway. Because the individuals of this species are so small direct observation of the calling behavior is very difficult and inaccurate. This study was undertaken to access the pattern of pheromone production in glands of virgin females as an indirect measure of the calling behavior. The major compound (5,9-dimethyl-pentadecane) was extracted to be used in two experiments. The first one investigated the effect of time of pheromone production by females. For this, extracts of 10 females of two days of age were done at 2-hour intervals during 24 hours (12:12 D:L). The other experiment evaluated the effect of female age in pheromone production. Age ranged from 1 to 5 days after emergence and 10 females of each age class were used. Extracts were made in hexane with 5ng/µl of (5,9-dimethyl-hexadecane) as internal standard and analyzed by GC. Females had the highest amount of pheromone at the last four hours in the dark and the two first hours in the light period. Amounts of pheromone were low in all other periods. One-day old females produced the highest amount of pheromone in the glands. After two days of emergence the titers of pheromone dropped significantly and remained low until females were 5 days old.

Financial Support: PNP&D - CAFÉ
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Universidade Federal de Viçosa, Núcleo de Biotecnologia Aplicada a Agricultura (BIOAGRO), 36571-000, Viçosa, MG, Brasil E-mail: mothman@insecta.ufv.br

Hiromi. Sasagawa¹, Christine Y. S. Peng², & *Shigeru Matuysma³.

The Oriental honey bee, Apis cerana (Ac), is known to be resistant to parasitic mite due to its hygienic grooming behavior. Such hygienic grooming behavior against Varroa jacobsoni has also been observed in the Japanese honey bee, Apis cerana japonica (Acj).

In the course of our study on the chemical ecology of honey bees in Asia, (R)-(-)-3-hydroxyoctanoic acid (3-HOA) was identified as forager specific mandibular gland components in Ac. In Acj, age and task related changes in worker mandibular acids were demonstrated. Workers younger than 18 days old produced 10-hydroxydecaoic acid (10-HDAA) and (2E)-10-hydroxydecenoic acid (10-HDA) as the major manbubular gland components (<5 micro g/bee). On the other hand, 21 days old workers and foragers were found to posses more than 10 micro g of 3-HOA as a main component.

In this study, biological activity of forager mandibular acids was tested. By using a conventional pollen germination test (10% sucrose-agar), ether extracts of both Acj and Am mandibular glands suppressed pollen germination completely at 1.0 head equivalent /ml and 0.5 head equivalent /ml and above respectively. Synthetic 3-HOA showed 100% suppression of pollen germination at 50 ppm (50ng/ml) and above. GC analyses after methylation by diazomethane showed that Acj foragers had an average of 28 micro g of 3-HOA/bee, while Am foragers possessed an average of 43 and 187 micro g of 10-HDAA and 10-HDA respectively.

3-Hydroxydecanoic acid (3-HDA, major) and 3-HOA (minor) were first identified from the mandibular glands of the leaf cutting ant (Atta sp.), and reported as an antifungal substance. Recently, we identified 3-HDA (major) and 3-HOA (minor) in the mandibular glands of several Bombus species in Japan. So far, 3-HOA was identified in forager mandibular gland of Apis cerana, collected from Japan, Korea, Taiwan, Vietnam, China, Nepal and Thailand. Other Apis species do not produce this acid. Distribution and biological function of these 3-hydroxy fatty acids among hymenopteran insects will be discussed.

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¹National Institute of Sericultural and Entomological Science, 1-2 Oowashi, Tsukuba. Ibaraki 305-8634, Japan.(Tokyo Metropolitan Inst. for Neuroscience, PRESTO JST), E-mail sasagawa@nises.affrc.go.jp; ² Department of Entomology, University of California-Davis, Davis, CA 95676, USA., and ³Inst. of Applied Biochem., Univ. of Tsukuba. Tsukuba. Ibaraki 305-8572, Japan.

We have shown that males of the butterfly Pieris napi transfer an anti-aphrodisiac to the females at mating, methyl-salicylate (Me-S), which the mated females emit when courted and makes males quickly abandon them (Andersson et al, 2000). Our recent results indicate that similar communication systems occur also in other pierid butterflies. However, it seems that the communication systems between the sexes is a theme with variations; in P. napi the anti-aphrodisiac consists of only one major substance, whereas the congener P. rapae seems to use two major components.

Andersson, J, Borg-Karlson A.K. and C. Wiklund. 2000. Sexual cooperation and conflict in butterflies: A male-transferred anti-aphrodisiac reduces harassment of recently mated females. Proceedings of the Royal Society of London B 267(July 7):1271.

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Johan Andersson Organic Chemistry Royal Institute of Technology

Teknikringen 56 plan 4 100 44 Stockholm _ Sweden

@-mail: joan@orgchem.kth.se

Josué Sant'Ana¹, Kathrin Stein¹, Arlene G. Corrêa^1, Evaldo F. Vilela², José R. P. Parra³, Jardel A. Moreira¹, Paulo C. Vieira¹, João B. Fernandes¹ Maria F. G. F. da Silva ¹ and Joseph C. Dickens⁴

Extracts of virgin females in supercritical fluid extraction (SFE) and also essential oil extracts from Citrus spp. (leaf, branch and orange peel) were recorded with a coupled gas chromatography-electroantennographic detection (GC-EAD) based on the antenna of both sex of the leafminer moth, Phyllocnistis citrella Station (Lepidoptera: Gracillariidae). The experiments in the CG-EAD with plant volatile revealed that two compounds from the whole blend of ether leaf, branch and orange peel are perceptible to males and females of the leaf miner moth. They have already been identified by gas chromatography-mass spectrometry (GC-MS) as linalool and decanal. A third minor compound present in the extracts of branch and orange peel also elicited an EAD response, but its structure remains unidentified. The males antennae responded to the females SFE, but no peaks in the extracts were detected by GC-MS. EAD profiles to 7Z,11Z- hexadecadienal, a known attractant to P. citrella, elicited a high EADs response from antennal receptors of co specific males. The bioassays revealed that the EAD antennal responses to SFE extracts coincided with the authentic 7Z,11Z- hexadecadienal, which indicates its function as a female-produced pheromone. Laboratory and field tests are going to be performed in order to elucidate the behavior response of P. citrella to the identified compounds.

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¹ UFSCar-DQ Laboratório de Síntese de Produtos Naturais-Via Washington Luís, km 235, CP: 676CEP: 13565-905 - São Carlos-SP, Brasil ; ² UFV- Departamento de Biologia Animal CEP: 36571-000- Viçosa-MG, Brasil; ³ ESALQ-USP _ Departamento de Entomologia - Piracicaba-SP, Brasil & ⁴USDA-ARS Plant Sciences Institute-Vegetable Laboratory-Bldg. 010A, Rm. 240 BARC-West- Beltsville, Maryland USA 20705