Screening tomato and cucurbit rootstocks for resistance to Verticillium dahliae*

Seedlings of tomato (19) and cucurbits (33), previously selected as rootstocks for commercial cultivars, were evaluated for their resistance to verticillium wilt under glasshouse conditions. Disease scoring was based on foliar symptoms, but in plants with no visual or ambiguous symptoms the presence of the pathogen was confirmed by isolations or PCR using specific DNA primers. Five of the cucurbit rootstocks showed resistance to infection, 11 were moderately infected, 11 were susceptible, while the remaining six were very susceptible. In general, Cucurbita pepo (courgette, pumpkin) and Lagenaria siceraria tolerated infection, Cucumis melo rootstocks were susceptible and Citrullus vulgaris was the most susceptible. When inoculated with race 1 of the pathogen, one of the tomato rootstocks was very resistant, three moderately resistant, nine tolerant, ten susceptible and one very susceptible. Pathogenicity tests with race 2 showed that none of the rootstocks exhibited high tolerance, but that there was some variation in susceptibility. Research is under way to evaluate the reaction of commercial cultivars to verticillium wilt when grafted onto resistant rootstocks.     

Progress in grafting watermelon to manage Verticillium wilt

Vegetable grafting for disease management was first used successfully when watermelon grafted onto a Cucurbita moschata rootstock overcame Fusarium wilt. Interspecific grafting has since been used effectively to mitigate several soilborne pathogens in a variety of solanaceous and cucurbitaceous cropping systems. Verticillium wilt caused by Verticillium dahliae is a significant disease in watermelon crops and is difficult to manage. Current management practices, including crop rotation, soil fumigation, and host resistance, are insufficient due to the ability of microsclerotia to persist in absence of a host, lack of efficacy of soil fumigants, and limited availability of resistant cultivars. Watermelon grafted onto commercial cucurbit rootstocks have increased tolerance to Verticillium wilt, although no cucurbit rootstocks are known to be completely resistant. Verticillium wilt incidence decreased on grafted plants grown in artificially and naturally infested soils, while scion health and growth as well as rootstock root mass and vigour increased. Commonly used rootstocks are Lagenaria siceraria, C. moschata, and C. maxima × C. moschata; of these, only C. maxima × C. moschata ‘Tetsukabuto’ reduced severity of Verticillium wilt across several scion cultivars, locations, years, and soil densities of V. dahliae. Although studies on Verticillium wilt resistance of grafted watermelon are few, their combined results suggest the threshold of V. dahliae soil density for watermelon may be around 5–12 cfu/g. This review summarizes available information on Verticillium wilt of watermelon and effects of different rootstock × scion combinations, assisting growers and breeding programmes in decisions to adopt watermelon grafting for management of Verticillium wilt.     

Distribution and multiplication of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in tomato plants with resistance derived from different origins

The distribution and multiplication of Ralstonia solanacearum in tomato plants of 11 resistant cultivars derived from different genetic sources and susceptible cultivar Ponderosa were examined. Bacterial multiplication in stems of resistant tomato plants was suppressed owing to the limitation of pathogen movement from the protoxylem or the primary xylem to other xylem tissues. The limitation was most conspicuous in Hawaii 7996. Grafting experiments indicated that the percentage of wilting of Ponderosa scions was less on Hawaii 7996 rootstocks than that on the most resistant rootstock (LS-89) used in Japan. Hawaii 7996 could be an alternative genetic source for breeding for resistance to bacterial wilt.     

Characterization of resistance against the olive‐defoliating Verticillium dahliae pathotype in selected clones of wild olive

Verticillium wilt of olive is best managed by resistant cultivars, but those currently available show incomplete resistance to the defoliating (D) Verticillium dahliae pathotype. Moreover, these cultivars do not satisfy consumers' demand for high yields and oil quality. Highly resistant rootstocks would be of paramount importance for production of agronomically adapted and commercially desirable olive cultivars in D V. dahliae‐infested soils. In this work, resistance to D V. dahliae in wild olive clones Ac‐13, Ac‐18, OutVert and StopVert was assessed by quantifying the fungal DNA along the stem using a highly sensitive real‐time quantitative polymerase chain reaction (qPCR) protocol and a stem colonization index (SCI) based on isolation of V. dahliae following artificial inoculations under conditions highly conducive for verticillium wilt. Ac‐13, Ac‐18, OutVert and StopVert showed a symptomless reaction to D V. dahliae. The mean amount of D V. dahliaeDNA quantified in stems of the four clones ranged from 3.64 to 28.89 pg/100 ng olive DNA, which was 249 to 1537 times lower than that in susceptible Picual olive. The reduction in the quantitative stem colonization of wild olive clones by D V. dahliae was also indicated by a sharp decrease in the SCI. Overall, there was a pattern of decreasing SCI in acropetal progression along the plant axis, as well as correlation between positive reisolation and quantification of pathogen DNA. The results of this research show that wild olive clones Ac‐13, Ac‐18, OutVert and StopVert have a valuable potential as rootstocks for the management of verticillium wilt in olive.     

Variability in infection and reproduction of Meloidogyne javanica on tomato rootstocks with the Mi resistance gene

The response of 10 commercial or experimental tomato rootstocks with the Mi resistance gene to an initial inoculum of a Mi‐avirulent population of Meloidogyne javanica was determined in pot tests conducted in spring and summer. In a field test, the rootstocks were subjected to continuous exposure to high initial population densities (2050 ± 900 second‐stage juveniles (J2) per 250 cm³ soil) of the nematode. The presence of the Mi locus in the resistant rootstocks and cultivars was confirmed using the PCR co‐dominant markers REX‐1 and Mi23. Nematode infectivity (egg masses) and reproduction (eggs g^?1 root) were highly variable in the spring tests. Rootstocks PG76, Gladiator and MKT‐410 consistently responded as highly resistant, with nematode multiplication rate (Pf/Pi) < 1 and reproduction index (RI) < 10%, and they were as efficient as standard resistant tomato cultivars at nematode suppression. The relative resistance levels of rootstocks Brigeor, 42851, 43965, Big Power and He‐Man varied depending on the susceptible standard used for reference or the duration of the test. Rootstocks Beaufort and Maxifort were susceptible to M. javanica (Pf/Pi > 50 and RI > 50%). Rootstocks PG76 and He‐Man, and the resistant tomato cv. Caramba showed high levels of resistance in the test conducted in summer, whereas MKT‐410 and 42851 and the resistant tomato cv. Monika were moderately resistant. In the field, seven rootstocks showed high levels of resistance and one (He‐Man) showed an intermediate level, whereas Beaufort and Maxifort were susceptible.     

Incidence and pathogenicity of races and isolates of Verticillium dahliae in Crete, southern Greece

Classification of 32 Verticillium dahliae isolates originating from 19 plant species in eight different botanical families to races and determination of host range pathogenicity were carried out. The physiological races of isolates were identified using the two differential tomato cultivars ??Belladonna?? (susceptible to both races 1 and 2 of V. dahliae) and ??Ace 55VF?? (resistant to race 1, susceptible to race 2 of V. dahliae). Among these isolates, 14 were race 2 (43.8%), 12 race 1 (37.5%) and six nonpathogenic (18.7%) on tomato. The host range pathogenicity of isolates was determined using four differential hosts (eggplant, turnip, tomato (Ve ^? ) and sweet pepper). Among isolates, five were pathogenic to both eggplant and turnip (15.6%), 21 to eggplant, turnip and tomato (65.6%), five to eggplant, turnip, tomato and sweet pepper (15.6%) and one was pathogenic to eggplant, turnip and sweet pepper (3.2%). The pathogenicity of isolates on the aforementioned five hosts was investigated on the basis of external symptoms and by calculating the relative areas under disease progress curves (relative AUDPC). Results showed that eggplant was the most susceptible, followed by turnip and tomato cv. Belladonna, while sweet pepper and tomato cv. Ace 55VF were less susceptible to all the isolates used. The pathogenicity of isolates varied from highly to mildly virulent on eggplant and turnip while on Belladonna, Ace 55VF and sweet pepper it varied from highly virulent to nonpathogenic. Belladonna exhibited a similar level of susceptibility to races 1 and 2 of V. dahliae, but was more susceptible than Ace 55VF to race 2. Interestingly, the isolates originating from eggplant were clearly more virulent than those originating from tomato and black nightshade on all solanaceous plants tested.     

Highly infested soils undermine the use of resistant olive rootstocks as a control method of verticillium wilt

Verticillium wilt of olive (VWO) is probably the most devastating fungal disease for olive trees worldwide, and currently the main cultivars are susceptible or moderately susceptible to this disease. The evaluation of resistant cultivars as rootstocks to control the disease has scarcely been explored, and mainly in short-term studies under controlled conditions, which usually do not correspond with field evaluations. The main objective of this study was to assess the responses to VWO of different scion × rootstock combinations of the olive cultivars Picual, Arbequina, Changlot Real, and Frantoio in a long-term field experiment with a soil highly infested with the defoliating pathotype of Verticillium dahliae. The results showed that grafting the susceptible cultivar Picual onto resistant rootstocks delayed the onset of the disease symptoms; however, after 4 years, it was observed that all combinations that contain Picual (a) were extensively colonized by V. dahliae; (b) developed severe symptoms of the disease; and (c) had plant mortality similar to Picual growing on its own roots. This result highlights the importance of long-term field experiments to evaluate VWO and shows that grafting susceptible olive cultivars onto resistant ones does not provide a durable control of VWO under high inoculum potential, as V. dahliae is able to progress through the resistant rootstock and then extensively colonize and kill the susceptible scion. However, the high inoculum potential observed in this study does not allow us to consider the evaluated resistant cultivars as completely ineffective under lower inoculum densities.     

Race 3, a new race of<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae </Emphasis>determined by a differential system with commercial cultivars

 Pathogenic variation among 26 Japanese isolates of Fusarium oxysporum f. sp. lactucae (FOL) was tested using 21 lettuce cultivars to select commercial lettuce cultivars as race differential indicators. Cultivar Costa Rica No. 4 was resistant to race 1 but susceptible to race 2, consistent with the conventional standard differential line VP1010. Cultivar Banchu Red Fire was susceptible to race 1 but resistant to race 2, which showed an opposite type of reaction as another differential line VP1013. Cultivar Patriot was susceptible to both races. The resistance reactions of the three cultivars under field conditions were identical with that observed in the seedlings. Thus cv. Costa Rica No. 4 and cv. Banchu Red Fire can be used as differential hosts to identify pathogenic races of FOL. This differential system showed that all FOL isolates obtained from diseased butterhead lettuce in Fukuoka, Japan were new races (i.e., pathogenic to three cultivars). We propose that the new race be designated race 3. Isolates of FOL, the pathogen of Fusarium wilt in lettuce, obtained from California showed the same reaction as that of race 1. Furthermore, the Japanese isolate SB1-1 (race 1) and California isolate HL-2 belonged to the same vegetative compatibility group. Our results suggest that both of the fungi are the same forma specialis. Received: March 25, 2002 / Accepted: August 26, 2002     

Occurrence of Fusarium oxysporum f. sp. melonis Race 1 in Japan

In 1994, Fusarium wilt of melon cultivars which are resistant to races 0 and 2 of Fusarium oxysporum f. sp. melonis was observed in southern area of the Lake Biwa region, Shiga prefecture. In commercial fields, mature plants of cv. Amus which were grafted onto cv. Enken Daigi 2, and of cv. FR Amus showed yellowing, wilting and finally death before harvesting of fruits. Diseased plants had vascular and root discolorations, and their stem sections yielded typical colonies of F. oxysporum. When the Shiga strains were tested for their pathogenicity to 12 species of cucurbits, they caused wilts only on melon. Using race differential cultivars of melon, the Shiga strains were classified as race 1 of F. oxysporum f. sp. melonis, which has not been reported in Japan. To further characterize their pathogenicity, the strains were used to inoculate 46 additional cultivars of melon, oriental melon and oriental pickling melon. All the race 1 strains were pathogenic to the cultivars tested, and their host range was apparently different from those of strains belonging to other races (races 0, 2 and 1,2y). DNA fingerprinting with a repetitive DNA sequence, FOLR3, differentiated race 1 strains from strains of races 0 and 2, but not from race 1,2y strains. Received 2 July 1999/ Accepted in revised form 30 September 1999     

Rootstock resistance to Fusarium wilt and effect on watermelon fruit yield and quality

The potential of grafted watermelon for resistance toFusarium oxysporum f.sp.niveum on some Curcurbitaceae,Lagenaria, Luffa, Benincasa and commercial rootstocks was evaluated. Effects of grafting on yield and quality of diseased plants were evaluated. All grafted plants and rootstocks were resistant to the three known races (0, 1, and 2) ofF. oxysporum f.sp.niveum except watermelon cv. ‘Crimson Tide’, which was susceptible to race 2. Fruit yield was positively (21–112%) affected byLagenaria rootstocks but negatively affected (200–267%) byCucurbita rootstocks when compared with the control. While only minor differences in fruit quality were determined in control and grafted plants onLagenaria rootstocks, the quality parameters for watermelon grafted ontoCucurbita rootstocks were lower than in the control. The reasons for low yield and quality might be due to an incompatibility betweenCucurbita rootstocks and watermelon. These results showed that rootstock influence on disease resistance as well as yield and quality of scion fruit is important in determining the potential use of grafting applications in watermelon. http://www.phytoparasitica.org posting Feb. 2, 2003.     

New virulent race of Phialophora gregata f. sp. adzukicola associated with continuous cultivation of adzuki bean cultivar Acc259

Adzuki bean cultivar Acc259, which is resistant to races 1 and 2 of Phialophora gregata f. sp. adzukicola, was used as a breeding resource for resistance to brown stem rot (BSR). During the third year after two successive cultivations of Acc259, a severe outbreak of BSR occurred in an experimental plot at the Tokachi Agricultural Experiment Station, Hokkaido, Japan. The isolates obtained from diseased plants were virulent to Erimo-shozu (susceptible to all races) and Acc259 but avirulent to Kita-no-otome (resistant to race 1 but susceptible to race 2). The existence of a new race of P. gregata f. sp. adzukicola, designated race 3, was determined; and its frequency in the plot soil was shown to increase from 16.7% before planting Acc259 to 100% after the third year. Of 140 isolates from the commercial production area that were formerly identified as race 1, 13 were actually race 3 and were restricted to certain limited fields.     

A New Race of Spinach Downy Mildew in Japan

A new race of spinach downy mildew caused by Peronospora effusa occurred in Fukui, Japan. The fungus was capable of affecting spinach cultivars resistant to races 1, 2, 3 and 4, but not some other cultivars. Thus, the fungus had different pathogenicity from race 3 and race 4 of the pathogen and was considered to be a new race of spinach downy mildew in Japan. Received 26 April 2001/ Accepted in revised form 17 August 2001     

Race distribution of Phytophthora sojae on soybean in Hyogo, Japan

Since 1987, Phytophthora root and stem rot of soybean [Glycine max (L.) Merr. cv. Tanbakuro], caused by Phytophthora sojae Kaufman and Gerdemann, has been increasing in the Sasayama, Nishiwaki, and Kasai regions in Hyogo, the most famous soybean (cv. Tanbakuro)-producing areas in Japan. In 2002 to 2004, 51 isolates (one from each field) of P. sojae were recovered from 51 fields in Hyogo. These isolates were tested for virulence on six Japanese differential soybean cultivars used for race determination in Japan, and three additional ones containing four Rps genes used in Indiana, USA. Race E was the most prevalent from 2002 to 2004, followed by races A, C, D, and four new races (proposed as races K, L, M, and N). Interestingly, none of the new races had high virulence on the Japanese differential cultivars, compared with other races in each area. One (race N) was avirulent on all six soybean differentials. There was a difference in race distribution on each of three individual areas; race E seemed to be a major component of the P. sojae population in Sasayama, whereas race A and the new race M were the most prevalent in Nishiwaki and Kasai, respectively. Rps6 (cv. Altona) and Rps1a + Rps7 (cv. Harosoy 63) were infected by 90.2% and 33.3% of all isolates, respectively. However, Rps1d (cv. PI103091) was not susceptible to any of the 51 isolates, nor was cv. Gedenshirazu-1. These two soybean cultivars were considered to be potential sources of resistance to breed new resistant cultivars with the desirable characteristics of cv. Tanbakuro for this region.     

Physiological Specialization of Fusarium oxysporum f. sp. lactucae, a Causal Organism of Fusarium Root Rot of Crisp Head Lettuce in Japan

In 1995, Fusarium root rot of crisp head lettuce, caused by Fusarium oxysporum f. sp. lactucae, was simultaneously found in the Shiojiri and Kawakami areas of Nagano Prefecture, Japan. The Shiojiri and Kawakami isolates differed in pathogenicity to lettuce cultivars. Because of this distinct physiological specialization, these Shiojiri and Kawakami isolates should be designated as race 1 and race 2, respectively, using lines VP1010 (highly resistant to race 1), VP1013 (highly resistant to race 2) and variety Patriot (highly susceptible to both races) as differential varieties. This is the first report of races of Fusarium oxysporum f. sp. lactucae, Received 21 September 2000/ Accepted in revised form 21 March 2001     

Pathogenic diversity within field populations of Orobanche cumana and different reactions on sunflower genotypes

Different races of the parasitic Orobanche cumana (sunflower broomrape) have been reported in Spain, race F being the most virulent. Full resistance in sunflower to races A–E is achieved with each of the single major genes Or₁ to Or₅ respectively. However, parasitised hybrids allegedly resistant to race F were observed in early 2002. The purpose of this study was to verify broomrape incidences (BI) on resistant sunflower genotypes, to assess the mixture of races within field populations and to test for partial resistance to race F in the sunflower hybrids showing a low degree of attack (DA) by the weed. Tests were conducted under field conditions in two locations of southern Spain. While no significant differences were found for yield and BI between locations, the DA on the cultivars depended on the location. With high infection levels and significantly lower yield in susceptible controls, marked differences in BI and DA were found within resistant cultivars, but all of them showed similar crop yield. When artificially inoculated with several populations of race F, line P96 and mainly line L86, were consistently slightly infected, suggesting they were inbred lines responsible for horizontal resistance in infested fields. L86 was extremely susceptible to race E populations, which is unusual as sunflower resistance to one race provided resistance to all the previously described races of O. cumana. No different virulences were detected within two groups of subpopulations (races E and F) inoculated onto resistant sunflower genotypes. However, race F subpopulations showed significant differences in aggressiveness, which seems to be related to horizontal (multigenic) resistance of the crop to the parasitic weed.     

Differentiation ofVerticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton

Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains ofVerticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars ofGossypium hirsutum, G. barbadense andG. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cotton-defoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data fromnit pairings indicated that the sub-populations ofV. dahliae (VCGs) may not be completely isolated genetically.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

Screening of cultivated and wild adzuki bean for resistance to race 3 of <Emphasis Type="Italic">Cadophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis>, cause of brown stem rot

Two diseases of adzuki bean, brown stem rot (BSR, caused by Cadophora gregata f. sp. adzukicola) and adzuki bean Fusarium wilt (AFW, caused by Fusarium oxysporum f. sp. adzukicola), are serious problems in Hokkaido and have been controlled using cultivars with multiple resistance. However, because a new race of BSR, designated race 3, was identified, sources of parental adzuki bean for resistance to race 3 were needed. Therefore, we examined 67 cultivars and lines of cultivated and wild adzuki bean maintained at the Tokachi Agricultural Experiment Station using a root-dip inoculation method. Consequently, nine adzuki bean cultivars, one wild adzuki bean accession and 30 lines (including two lines resistant to all the three races of BSR and AFW) were confirmed to be resistant or tolerant to race 3 of BSR, and we found a cultivar Akamame as well as a wild adzuki bean Acc2515 to be a new source for a resistance gene to the race 3. This cultivar also holds promise as a source of resistance against other races of BSR and AFW.     

Physiologic races ofFusarium oxysporum f.sp.melonis in the southeastern anatolia region of turkey and varietal reactions to races of the pathogen

Thirty-four isolates ofFusarium oxysporum f.sp.melonis (F.o.m.) obtained from 205 fields in melon-producing areas in the southeastern Anatolia Region of Turkey were identified on the basis of colony morphology and pathogenicity by the root dip method. In this region the mean prevalence of wilt disease was 88.1% and the mean incidence of disease was 47.5%. Physiologic races 0, 1, 2, and 1,2 of the pathogen were determined by their reactions on differential melon cultivars ‘Charentais T,’ ‘Isoblon’, ‘Isovac’ and ‘Margot’ in the greenhouse. Race 1,2, representating 58.8% (20/34) of all isolates, was widely distributed. Of the other pathogenic isolates, eight were identified as race 0, five as race 1, and one as race 2. This is the first report of physiologic races ofF.o.m. in Turkey. Of 44 melon cultivars tested in the greenhouse for resistance toF.o.m. races, 36 were found to be moderately resistant to race 0, 17 were susceptible to race 1,2, 34.1% were highly resistant to race 1, and 52.2% had moderate resistance to race 2. http://www.phytoparasitica.org posting July 16, 2002.     

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in resistant and susceptible wild olive clones

Verticillium wilt (VW) in olive is best managed by an integrated disease management strategy, of which use of host resistance is a key element. The widespread occurrence of a highly virulent defoliating (D) Verticillium dahliae pathotype has jeopardized the use of commercial olive cultivars lacking sufficient resistance to this pathogen. However, the combined use of resistant wild olive rootstocks and Trichoderma spp. effective in the biocontrol of VW can improve the management of VW in olive. In vivo interactions between D V. dahliae and Trichoderma harzianum were studied in olive and wild olive plants displaying different degrees of resistance against this pathogen using confocal microscopy. This multitrophic system included wild olive clones Ac‐4 and Ac‐15, olive cv. Picual, and the fungal fluorescent transformants T. harzianum GFP22 and V. dahliae V138I‐YFP, the latter being obtained in this study. In planta observations indicated that V138I‐YFP colonizes the roots and stems of the olive and wild olive genotypes, and that GFP22 grows endophytically within the roots of them all. YFP fluorescence signal quantifications showed that: (i) the degree of root and stem colonization by the pathogen varied depending upon the susceptibility of the tested wild olive genotype, being higher in Ac‐15 than in Ac‐4 plants; and (ii) treatment with T. harzianum GFP22 reduced the extent of pathogen growth in both clones. Moreover, root colonization by strain GFP22 reduced the percentage of pathogen colonies recovered from stems of olive and wild olive plants.