Natural Products for Pest Management - American Chemical Society

Chapter 7

Search for Germination Stimulants and Inhibitors for Root Parasitic Weeds

Downloaded by FUDAN UNIV on April 24, 2017 | http://pubs.acs.org Publication Date: May 25, 2006 | doi: 10.1021/bk-2006-0927.ch007

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Koichi Yoneyama , Daisuke Sato , Yasutomo Takeuchi , Hitoshi Sekimoto , Takao Yokota , and Takashi Sassa 2

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Center for Research on Wild Plants and Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan Department of Biosciences, Teikyo University, Utsunomiya 320-8511, Japan Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan 3

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Germination of root parasitic weeds Striga and Orobanche is induced only after an exposure to stimulants in root exudates of host and some non-host plants. Since the isolation and characterization of strigol as the first Striga germination stimulant, five strigol-related compounds, termed strigolactones, have been isolated and characterized as natural stimulants. However, plants seem to produce many other stimulants including novel strigolactones. In addition to these natural stimulants, fungal metabolites, cotylenins and fuscicoccins, and plant hormone jasmonate and its analogues were found to elicit germination of these root parasites. In this paper, characterization and structure-activity relationships of these germination stimulants and other fungal metabolites as germination inhibitors are discussed.

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© 2006 American Chemical Society Rimando and Duke; Natural Products for Pest Management ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


89 Among the parasitic angiosperms, witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are the two most devastating weeds on several cereal and leguminous crops, respectively. These parasites produce large numbers of tiny seeds with prolonged viability and special germination requirements. To germinate, the seeds should be kept in a warm moist environment for several days (termed conditioning) and subsequently be exposed to exogenous germination stimulants (7). Once germinated, these parasites will die within a week unless they attach to the roots of host plants. Therefore, inducing seed germination in the absence of host plants, termed "suicidal germination", is a promising strategy for depleting seed reserves in soil (2). For this purpose, germination stimulants that are stable under field conditions should be found or developed, because natural germination stimulants identified so far are rather unstable and decompose rapidly in soil. On the other hand, compounds that inhibit germination of these parasites without causing damage to host plants and to the environment are of practical importance, since those compounds may be used in the heavily-infested fields where crops susceptible to the parasites are to be planted. In this report, occurrence and structures of natural and false germination stimulants for root parasites, Striga and Orobanche, are presented along with inhibitors of germination.

Germination Stimulants It should be emphasized that conditioned seeds of root parasites Striga and Orobanche will not germinate unless they are exposed to exogenous germination stimulants. There are some reports on "spontaneous germination" in the absence of such a stimulant, but most of them were not reproducible. It is likely that, under natural conditions, a portion of parasite seed population in soil are conditioned and only a portion of them receive germination stimulants. Since some of these root parasites parasitize a wide range of plant species, many plant species produce and release germination stimulants for these parasites. In addition, as discussed later, some microorganisms were found to produce chemicals that elicit parasite seed germination as well as other chemicals that inhibit germination. Therefore, germination rates of these parasites in soil might be relatively high. In this paper, these germination stimulants are classified into two groups; natural germination stimulants which play important roles in host recognition by root parasites and false germination stimulants that are not involved parasite-host interactions and thus may be used as suicidal germination stimulants.

Rimando and Duke; Natural Products for Pest Management ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Natural Germination Stimulants; Strigolactones A group of sesquiterpene lactones, collectively called strigolactones (Figure 1) are potent germination stimulants for both Striga and Orobanche. To date, 5 natural strigolactones have been characterized. They are strigol, strigyl acetate, sorgolactone, alectrol (tentative structure), and orobanchol. Strigol and strigyl acetate, the first strigolactones, were isolated from root exudates of a false host cotton (3, 4). Later, Siame et al. identified strigol in the root exudates of genuine hosts of Striga, sorghum, maize, and proso millet (5). Therefore, strigol is produced by both host and non-host plants. B y contrast, all of the other natural strigolactones have been isolated from host plants; sorgolactone from sorghum (6), alectrol from cowpea (7), and orobanchol from red clover (8). It is likely, however, that these strigolactones are also produced by non-host plants.

alectrol (tentative structure)

sorgolactone

Figure 1. Chemical structures of natural strigolactones.

In the search for natural germination stimulants, various hosts of Striga or Orobanche were grown hydroponically and root exudates were collected weekly. These root exudates were extracted with ethyl acetate, and the ethyl acetate extracts were examined for germination stimulation activity on O. minor Sm. The results are summarized in Figure 2 in which germination rates o f the seeds treated with a portion of the ethyl acetate extracts corresponding to 1 mL, 5 mL or 20 mL of the root exudates are shown. In Figure 2, the number of plants, their growth stages, and growth media were optimized to each plant species, and thus direct comparison of germination stimulation activity between different plant species is not applicable. Most of plant species examined were found to produce


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germination stimulants immediately after germination. However, for example, the root exudates of young tomato seedlings (~3 week-old), a host of Orobanche, did not induce germination at all. Furthermore, nutrients influenced production of germination stimulants and their effects varied with plant species. Since most of the plants grown hydroponically produced germination stimulants, crude extracts of root exudates were purified by reverse phase (ODS) H P L C . The fractions obtained were tested for germination stimulation on O. minor.

mL eq. mL eq. O20 mL eq. • 1 B 5

40 60 Germination (%)

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Figure 2. Germination stimulation on Orobanche minor by root exudates of various crop plants.

In the case of soybean, for example, there were two active fractions after H P L C separation of ethyl acetate extracts of root exudates and these fractions seemed to correspond to orobanchol and alectrol based on their retention times. The same sample was then analyzed by HPLC-tandem mass spectrometry ( L C / M S / M S ) using multiple reaction monitoring ( M R M ) method. For M R M , transition of m/z 369 > 272 corresponding to [ M + N a ] and [ M + N a - D ring] , respectively, was monitored to detect strigol and its isomers including orobanchol and alectrol (9, 10). In the M R M chromatograms shown in Figure 3 +


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Figure 3. MRM chromatograms of soybean root exudates grown under low nitrogen (upper, 1/10 N) and normal conditions (lower).



93 (clear in the upper chromatogram), two peaks were detected at retention times of 11.3 and 25.6 min, corresponding to orobanchol and alectrol, respectively. The effect of nutrients (nitrogen) in the growth media (basal medium was Tadano and Tanno medium) on stimulant production was evident; soybean plants produced larger amounts of germination stimulants when grown under low (1/10) N conditions (6.8 ppm N , upper chromatogram) as compared to the normal N conditions (68 ppm N , lower chromatogram). These results clearly indicated that soybean produced two known strigolactones, orobanchol and alectrol, but not strigol, and their productions seemed to be promoted under low nitrogen availability as in the case of red clover (11). The distribution profiles of germination stimulation activity after ODSH P L C demonstrated that some of host plants produced novel stimulants as listed in Table I. Among these novel stimulants, there are at least one strigol isomer, two dehydro-strigol isomers, and four tetradehydro-strigol isomers.

False Germination Stimulants; Fungal Metabolites and Jasmonates Natural germination stimulants such as strigolactones and dihydrosorgoleone are unstable in soil, and no useful and economic suicidal germination stimulants based on these molecules have been obtained, except for promising stimulants developed by Zwanenburg's group at Nijmegen University, the Netherlands (72). Germination stimulants structurally unrelated to the natural ones may be important molecular probes to understand the germination mechanism of the parasites, and also to develop useful and economic germination stimulants. Striga seed germination is also stimulated by other chemicals including natural and synthetic cytokinins, scopoletin, inositol, methionine, sodium hypochlorite, and ethylene. Strigolactones were reported to induce ethylene biosynthesis in Striga seeds, and both ethylene biosynthesis and action are required for germination. Therefore, any compounds which stimulate ethylene production may induce Striga germination. In fact, 1-aminocyclopropane-lcarboxylate (ACC), the immediate biosynthetic precursor of ethylene, is a good germination inducer for Striga seeds. However, ethylene and A C C seem to not be active on Orobanche germination (73).

Cotylenins and Fusicoccins Screening of microbial and fungal metabolites for activity as Striga and Orobanche germination stimulants is probably a more promising strategy. In fact, several fungal metabolites were found to induce Striga and Orobanche germination. In particular, cotylenins (CNs) and fusicoccins (FCs) produced by Cladosporium sp. 501-7W (14, 15) and Fusicoccum amygdalie Del. (16, 17),


94 Table I. Germination Stimulants Produced by Various Host Plants Plant species Red clover Soybean Cotton Carrot Tomato Pea Sorghum Maize

Germination Stimulants Orobanchol, alectrol, unknown Orobanchol, alectrol Strigol, strigyl acetate Novel strigolactones Novel strigolactones Novel strigolactones Novel strigolactones, sorgolactone (strigol) Novel strigolactones, sorgolactone


Note: Production of strigolactones, except for strigol in sorghum, was confirmed byLC/MS/MS. 5

respectively, elicited both Striga and Orobanche seed germination at < 10" M (18). Structures of cotylenin A and fusicoccin A are shown in Figure 4. Since the structures of CNs and FCs are totally different from those of strigolactones, and, in addition, these fungal metabolites are 1000 to 10000 times less active than strigolactones, they may elicit seed germination by a mechanism different from that by strigolactones. However, in the case of S. hermonthica seed germination, both C N - and strigol-induced germinations were reduced by inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, A V G ) and action (sodium thiosulfate, STS), indicating that ethylene is involved in both cases (18).

cotylenin A

ilisicoccinA

Figure 4. Structures of cotylenin A andfusicoccin A.

Natural and synthetic derivatives of CNs and FCs, fusicoccanes, were then examined to clarify structure-activity relationships in germination stimulation on O. minor seeds. In general, structural requirements for parasite germination were similar to those for lettuce seed germination. The substituent on C (Ri) is not necessary for activity and can be H , O H , or an Osugar group (see Figure 5). The substituent on C (R ) may be needed for 9-deoxy derivatives. The substituent on C (R3) should be a hydroxyl group for derivatives lacking 3-OH (R4=H) and vice versa. Although germination stimulation activities of CNs, 9

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95 FCs, and their analogues are weak, these compounds, in particular, those lacking 3-OH (FC type compounds), are much more stable than strigolactones in soil, and therefore appropriate structural modifications may afford useful suicidal germination inducers for root parasites (19). In general, Orobanche spp. appeared to have stricter germination requirements than Striga spp., and the compounds which elicited Orobanche germination also induced Striga germination. Except for CNs and FCs, only strigolactones have been reported to induce Orobanche germination (7). In contrast, in addition to CNs, FCs, and strigolactones, Striga germination is induced by other chemicals including ethylene, cytokinins, and auxins as mentioned before. Although various plant growth regulators (gibberellins, cytokinins, auxins, abscisic acid, ethylene, and brassinosteroids) have been found not to induce Orobanche germination, jasmonate (JA) had not been examined for its effect on the seed germination of these root parasites. Therefore, J A was included in the assay and found to induce the germination of O. minor (20). J A and related compounds were then examined for their effects on the seed germination of O. minor and S. hermonthica. Among the compounds examined, esters were more active than the corresponding free acids, and methyl jasmonate (MJA) and 6-ep/-9,10dihydrocucurbate were the two most active stimulants (Figure 6). M J A induced more than 50% germination of O. minor and 5. hermonthica seeds at 10" M . Unfortunately, M J A , JA, and its related compounds are not stable in soil. 4

Germination Inhibitors Specific inhibitors of parasite seed germination are useful molecular tools to unveil germination mechanisms. In addition, these inhibitors may be used in the field to reduce germination of parasite seeds i f they are stable and safe enough in the environment. Unfortunately, there have been no reports on specific inhibitors of parasite seed germination. Some fimgal metabolites, however, inhibit Striga and Orobanche germination stronger than those of crop seeds such as lettuce and sorghum. For example, two fungal metabolites, 4,15-diacetoxyscirpenol and 4,15diacetylnivalenol were isolated as potent germination inhibitors from fungi, probably Fusarium spp., contaminated O. minor seeds (Figure 7). These compounds inhibited O. minor seed germination at < 10~ M without affecting germination and growth of lettuce and maize. Zonno et al. reported Striga and Orobanche germination inhibition by fungal toxins (21, 22). These mycotoxins are highly toxic to mammals and thus these compounds cannot be used as seed killers. However, these toxins may be important as active principles produced by Fusarium spp., promising biological control agents of Striga and Orobanche (23-25). 5



Figure 5. General structures offusicoccanes.

Figure 6. Two most active jasmonate-type germination stimulants.


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t>Ac AcO

)Ac

4,15-diacetoxyscirpenol

4,15-diacetylnivalenol

Figure 7. Fungal metabolites isolated as germination inhibitors for root parasites.


Conclusion Various plant species have been shown to produce and release germination stimulants for root parasites. Among these germination stimulants, more than 10 compounds are confirmed to be strigolactones by L C / M S / M S analyses, indicating that strigolactones are distributed widely in the plant kingdom. In addition, most host plants examined were found to produce more than one stimulant, and thus these plants may produce individual stimulants at different levels under different growth conditions and/or different growth stages. Once germination stimulants are characterized, chemical analyses like L C / M S / M S may help in screening resistant cultivars for low stimulant production or trap crops for high stimulant production. In addition, some cultivars may be resistant because they produce not only stimulants but also specific inhibitors of germination.

Acknowledgment Part of the study was supported by Grants-in-Aid for Scientific Research (12460049, 12556015, 15380079) from the Japanese Society for the Promotion of Science (JSPS) and Priority Research Projects, Utsunomiya University.

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