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Germination ecology, emergence and host detection in Cuscuta campestris

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Trials were carried out to study the germination and dormancy of Cuscuta campestris Y. (dodder) seeds and factors influencing the success of early parasitisation of sugarbeet. Primary dormancy can be removed by seed scarification. Germination was
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  Germination ecology, emergence and host detectionin  Cuscuta campestris  S BENVENUTI*, G DINELLI  , A BONETTI   & P CATIZONE  * Department of Agronomy and Agroecosystems Management, University of Pisa, Pisa, Italy and    Department of Agroenvironmental Scienceand Technology, University of Bologna, Bologna, Italy Received 29 April 2004Revised version accepted 28 January 2005 Summary Trials were carried out to study the germination anddormancy of   Cuscuta campestris  Y. (dodder) seeds andfactors influencing the success of early parasitisation of sugarbeet. Primary dormancy can be removed by seedscarification. Germination was negligible at 10  C andoptimalat30  C,whileitwasnotinfluencedbylight.Seedburial induced a cycle of induction and breaking of secondary dormancy. Seedling emergence was inverselyproportional to the depth of seed burial and only seedburiedwithin5 cmofthesoilsurfaceemerged.Storageof  C.campestris seedsinalaboratoryfor12 yearsresultedinthe loss of primary dormancy, enabling the germinationof all viable seeds. Host infection (i.e. protrusion of parasite haustoria from host tissue) was heavily influ-enced by host growth stage. Tropism towards a host wasduetotheperceptionoflighttransmittedbygreenpartsof sugarbeet plants. Insertion of a transparent glass sheetbetweenhostleavesandparasiteseedlingsdidnotmodifythis response. This phototropism permitted  Cuscuta  toidentify host plants with high chlorophyll content as afunctionofthelowerred/farredratiooftransmittedlight. Keywords:  Cuscuta campestris , parasitic weed, seedgermination, seed dormancy, phototropism.B ENVENUTI  S, D INELLI  G, B ONETTI  A & C ATIZONE  P (2005) Germination ecology, emergence and host detection in Cuscuta campestris .  Weed Research  45 , 270–278. Introduction Cuscuta campestris  Yuncker (dodder), a parasitic weedbelonging to the Convolvulaceae family, is widespreadboth in temperate and subtropical ecosystems (Dawson et al. ,1994).Itparasitisesseveralcropsandweeds(Parker& Riches, 1993; Holm  et al. , 1997). Monocotyledons arerarelyattacked(Haidair et al. ,1997),probablybecauseof the low efficacy of   C. campestris  enzymes involved in thelysis of monocotyledon tissues during penetration by theparasite. Control of this weed is particularly difficult insugarbeet and lucerne in temperate agriculture (Cooke &Black,1987;Crooker,1987).Chemicalcontrolisdifficult,becauseofthelackofselectiveherbicides(Dawson,1990)and because of its tolerance to broad-spectrum products,such as glyphosate (Nadler-Hassar & Rubin, 2003).An important trait ensuring the success of  C. campestris  as a crop parasite is seed dormancy(Hutchinson & Ashton, 1980). Dormancy influencesgermination dynamics, permitting late-emerging plantsto escape control practices, including cultivation andherbicide application. Unlike the important hemi-para-sitic weeds in the genera  Orobanche  or  Striga ,  Cuscuta spp. do not require host-root exudates to stimulategermination (Vail  et al. , 1990; Benvenuti  et al. , 2002).Dormancy is due to the presence of a hard seed coat(Lyshede, 1992). This physical dormancy remains untilsoil microorganism activity or tillage cause scarificationof the seed coat. This characteristic makes the seedsparticularly resistant even to soil solarization (Haidar et al. , 1999). It is not clear if secondary dormancy,typical of the germination cycle of many weed species(Bouwmeester & Karssen, 1989), is present in  Cuscuta spp. Little information is available on seed longevity,ability to emerge from different burial depths andviability after long-term storage, but it is clear thatspring germination is strongly influenced by temperature(Allred & Tingey, 1964). Correspondence : S Benvenuti, Department of Agronomy and Agroecosystems Management, University of Pisa, Via S. Michele 2, 56124, Pisa, Italy.Tel: (+39) 050 599111; Fax: (+39) 050 540633; E-mail: sbenve@agr.unipi.it   2005 European Weed Research Society  Weed Research  2005  45,  270–278  After germination, seedlings of   Cuscuta  spp. under-go a non-parasitic phase of growth, dependent on seedreserves, for 2–3 weeks. Trophic growth of the shootapex towards a host, probably induced by a low fluenceresponse (LFR) (Hartmann, 1966), is controlled byphytochrome (Kujasky & Truscott, 1974). Indeed,tissues of   Cuscuta  plants have a low chlorophyllcontent (MacLeod, 1961), including low accessoryphotosynthetic pigments such as alpha- and beta-carotenes and xanthophylls (Dinelli  et al. , 1993). Likeother holoparasites,  Cuscuta  plants lack leaves androots, with sugars, water and minerals absorbeddirectly from host phloem and xylem tissues (Zimmer-mann, 1962; Press  et al. , 1990). Host recognition ismediated by a phototropic mechanism (Haidair  et al. ,1997). Both chemiotropism and tigmotropism (Bu ¨nning& Kautt, 1956; Jaffe, 1973) were implicated in hostperception, but neither has been proven. Seedlings of the heterotrophic species  Cuscuta planiflora  Ten., growin the direction of far-red light (Orr  et al. , 1996). Thisresponse appears to have evolved to improve thechances of   Cuscuta  spp. detecting a host in leaf canopies of natural habitats. Indeed, far-red lightindicates the presence of vegetation, as chlorophyllabsorbs light in the red region and transmits it in thefar-red (Holmes & Smith, 1975).In spite of this knowledge, several aspects of thegermination and early growth of   C. campestris  are stillunclear, including maximum host–weed distance forparasitisation, importance of the phenological stage of the host and the mechanism of host detection. The aimof this study was to investigate the primary andsecondary dormancy, emergence from different burialdepths and germination characteristics of   C. campestris seed after long-term storage. A further objective was toinvestigate parasitisation during the first growth stagesof   Cuscuta , as a function of host distance, phenologicalstage and optical characteristics of host leaves. Materials and methods Materials  Cuscuta campestris  seeds were collected at the end of August, 1990, from plants parasitising sugarbeet ( Betavulgaris  var.  saccharata  L.) in the Po Valley (Malacappa,Bologna, Italy). Seeds were collected from maturecapsules and subsequently dried in a greenhouse for1 month. Dried seeds were stored in two ways: (i) in acontrolled chamber with constant humidity and tem-perature [20  C and 60% relative humidity (RH)]; (ii)immediately buried 25-cm deep in loam soil at theUniversity of Bologna experimental farm (CadrianoBologna, Italy) in permeable plastic bags with a finemesh of 500  l m. Soil moisture content was high duringthe winter and often near field capacity. Germination  A month after collection, germination tests were per-formed to assess primary dormancy. Fifty seeds (laborat-orystored)wereplacedin9-cmPetridisheswithfilterpapermoistened with distilled water to permit seed imbibition.Half of each seed batch was scarified by immersion for10 min in concentrated sulphuric acid. Incubation wasperformedinaclimatechamberataconstanttemperaturefor three weeks either in light (Philips THL 20W/33fluorescentneon; 100  l E m ) 2 s ) 1 , Philips,Eindhoven,theNetherlands) or in darkness. Seeds were consideredgerminated after radicle protrusion (2–3 mm). Germination dynamics  During the first 2 years after seed collection, germina-tion tests were performed every 4 months to investigateinduction and the breaking of secondary dormancy.These tests were carried out with laboratory- and field-stored seeds. For each storage time, seeds were placed onmoistened sheets of filter paper in Petri dishes andincubated in a growth chamber at the near-optimaltemperature of 30  C (see germination results) with a12-h photoperiod. Emergence  Tests were performed in plastic pots (12 cm  · 12 cm  ·  10 cm) perforated at the base to ensure optimalsubstrate humidity and to permit sub-irrigation. Potswere filled with loam soil (collected at the CadrianoExperimental Farm of the University of Bologna). Thesoilwascollectedinanareanotinfestedby C. campestris ,in order to avoid any interference in the determination of emergence. Fifty seeds (both laboratory- and field-storedfor 16 months) were sown in each pot at depths of 0, 0.5,1, 2, 4, 6, 8 cm (only one depth for each pot). Pots werefilled with an equal amount (by weight) of soil andcompacted uniformly. After sowing, pots were sub-irrigatedandsoilmaintainedatapproximatelyhalfofthewater field capacity (WFC). Pots were incubated in agrowth chamber at 30  C with a 12-h photoperiod.Emergence was determined every day for 2 weeks,removing seedlings after emergence. Germination and emergence rate  The mean germination time (MGT) in Petri dishes andthe mean emergence time (MET) in soil was calculatedaccording to the following formula: Cuscuta  seed germination and host detection  271   2005 European Weed Research Society  Weed Research  2005  45,  270–278  MGT or MET ¼ X n  g N  where  n  is number of germinated seeds or emergedseedlings on day  g  and  N   is the total number of germinatedseeds or emerged seedlings. Parasitisation  Parasitisation tests were carried out using sugarbeet as ahost.Sugarbeetseedswereplacedinplasticpots(10 cm  · 14 cm  ·  8 cm) filled with the above-cited soil mixed withpeat (80% loam soil, 20% peat). After germination,sugarbeet seedlings were thinned to three plants per pot.Pre-germinated  C. campestris  seeds were sown in to pots(50 each) either without hosts or in the presence of sugarbeet plants at the four-, or six- to eight-leaf stage.The following parameters were determined: (i) totalnumber of   C. campestris  seedlings reaching (weed-cropcontact) the host; (ii) total number of   C. campestris seedlings parasitising the host. Parasitisation was consid-ered successful when haustoria penetrated the hosttissues. Phototropic tests  Germinated  C. campestris  seeds with a 1–2-mm-longtendril were placed on imbibed filter paper (WhatmanNo. 3) inside black plastic boxes (10 cm  ·  10 cm  · 10 cm) without covers. Boxes were irradiated withwhite light (100  l E m ) 2 s ) 1 ) using a fluorescent neonbulb (Philips TLF 20W/33). Circular windows (5 cmdiameter) were made on two opposite sides of eachbox. In one window, a sugarbeet leaf characterized byhigh chlorophyll content was attached and sealed withblack adhesive tape in order to permit only the entryof leaf-transmitted light. In the other window, asugarbeet leaf characterized by low chlorophyll con-tent was attached. The chlorophyll content of thesugarbeet leaves was determined according to themethod reported below. Outside each window, incan-descent lamps (50  l E m ) 2 s ) 1 ; OSRAM 100 W) wereplaced. Between the windows and the incandescentlamps, water filters (transparent glass boxes filled withwater, 5 cm  ·  10 cm  ·  10 cm) were inserted in orderto stop infra-red rays and to avoid the heating of parasite seedlings. Light intensity transmitted bysugarbeet leaves and reaching  C. campestris  seedlingswas approximately 5  l E m ) 2 s ) 1 . Sugarbeet leaveswere changed daily to maintain their optical charac-teristics. Boxes were placed in temperature-controlledcabinets at 25  C that also prevented interference fromother light sources. Analysis of chlorophyll and quality of transmitted light  Sugarbeet leaves (phenological stage of rosette, fromplants about 4 months old) for phototropic tests werecollected from a field near Pisa (July 2003). Dark greenand light green leaves were chosen and two groups of leaves were formed according to their chlorophyllcontent. One-centimetre-diameter leaf discs were ana-lysed for their chlorophyll  a  and  b  content according toMoran (1982) and chlorophyll  a  and  b  absorbance wasmeasured by a spectrophotometer (Shimadzu Mod.UV-1204, Shimadzu Corporation, Tokyo, Japan). Thehigh-chlorophyll leaves were characterized by a totalchlorophyll content 10% higher with respect to themean value of all sugarbeet leaves sampled. The lowchlorophyll leaves chosen for phototropic tests werecharacterized by a total chlorophyll content 10% lowerthan the mean value of all sugarbeet leaves sampled. Theintensity of transmitted light and the related-red/far-redratio (irradiance at 660 and 730 nm) were measured by aspectroradiometer (LI-COR 1800, Lincoln, NE, USA). Seed longevity  Cuscuta campestris  seed germination was assessed after12 years of laboratory storage at constant humidity andtemperature (60% RH at 20  C). Intact and sulphuricacid-scarified seeds were incubated at 30  C with a 12-hphotoperiod. The tetrazolium test (ISTA, 1999) was alsoperformed to investigate seed viability. Statistical analysis  All experiments had three to four replicates and wererepeated twice. Data were pooled over the two experi-ments because there was no interaction. A completelyrandomized experimental design was adopted for ger-mination, emergence and parasitisation tests. Aftertesting for homogeneity of variance, all per cent datawere arcsin-transformed. Angular values were subjectedto analysis of variance ( ANOVA ) with mean separation bythe Student–Newman–Keul’s test. Differences wereaccepted as significant at  P  < 0.05. CoStat software(CoHort, Minneapolis, MN, USA) was employed foreach statistical analysis. Results and discussion Germination tests  Germination of fresh  C. campestris  seeds (intact orscarified), as a function of light and temperature 272  S Benvenuti  et al.   2005 European Weed Research Society  Weed Research  2005  45,  270–278  conditions, is reported in Fig. 1. As a consequence of ahard seed coat, a scarification treatment was crucial forthe induction of germination. Scarification significantlyincreased germination in both seed samples ( P  £  0.05).Germination of intact seeds did not exceed 20%, whilein the temperature range from 20 to 35  C, germinationafter scarification was greater than 80%. It is importantto note that the selected scarification time (10 min) wasvirtually optimal as a longer treatment time did notincrease germination (data not shown). Cuscuta campestris  germination was strongly depend-ent on incubation temperature. Below 15  C, germinationwas negligible and with increasing temperature, germi-nation of both intact and scarified seeds increased up to30  C. Above 30  C, a slight decrease in germination wasobserved. This requirement of high temperatures foroptimal germination, appears to be a survival strategy of  C. campestris . Indeed, the need to germinate only in thepresence of existing vegetation could have induced theselection of genotypes capable of colonizing crops in latespringand/orsummer,whenthetemperaturesarehigher.Seed germination was not influenced by light, con-firming the photo-insensitive germination ecology of  Cuscuta  (Hutchinson & Ashton, 1980). In addition, thegermination of photoblastic seed is generally inhibitedby low-red/far-red ratios, which are usually presentunder a crop canopies (Taylorson, 1969). However C. campestris  does not need to block germination toavoid competition, as the presence of the host isindispensable for its survival. The requirement of lightfor germination is often linked to the presence of chlorophyll in floral tissues of the mother plant, aschlorophyll modifies the quality of light transmitted tothe seeds by converting phytochrome to the inactiveform (Cresswel & Grime, 1981). This phenomenon is notseen in  C. campestris  which lacks chlorophyll. Dormancy dynamics  The germination of laboratory-stored or buried seeds atvarious intervals during a 2-year period after collectionis shown in Fig. 2. Germination of laboratory-storedseed did not vary significantly, remaining at about 10%for the entire period. In contrast, field storage caused aprogressive reduction of primary dormancy. After4 months, germination doubled (25%) and after8 months it reached 60%. In soil the action of abiotic(heat and water stress) and biotic (microorganisms)factors causes the scarification of the seed coat and thebreaking of primary dormancy. For many species,primary dormancy declines after seed dispersal (Hil-horst, 1995). However, after 1 year of field storage,germination decreased to 10% and then increased toaround 70–75% after 16–21 months. Finally, after2 years, buried seeds were again largely dormant,exhibiting a germination value of approximately 10%.The results obtained provide evidence of the inductionand breaking of secondary dormancy in  C. campestris , aphenomenon already observed in several species presentin agro-ecosystems (Bouwmeester & Karssen, 1989) andin natural ecosystems (Baskin & Baskin, 1985). In C. campestris , secondary dormancy is induced at the endof summer to prevent germination during autumn/winter to avoid low temperatures, when potential hostsin temperate areas are in short supply. The breaking of secondary dormancy occurs at the end of the winterwhen temperatures are increasing and conditions forgermination and growth of hosts are favourable. Even if physiological causes for the induction and breaking of secondary dormancy are still unclear, it has beenhypothesized that the fluidity of membranes (Hilhorst,1998) could play a central role in inducing or blocking 051015202530350102030405060708090100 LightDark Scarified seedsIntact seeds Temperature (°C)    G  e  r  m   i  n  a   t   i  o  n   (   %   ) Fig. 1  Germination of fresh intact or scarified  C. campestris  seedsincubated at increasing temperatures (from 5 to 35  C) in light ordark conditions. Vertical bars indicate standard errors of the meanvalues. 0510152025020406080100 Laboratory storageField storage Time after seed collection (month)    G  e  r  m   i  n  a   t   i  o  n   (   %   ) Fig. 2  Time trend germination of laboratory- ( d - d ) and field-stored ( s - s )  C. campestris  seeds. Seeds were incubated in a growthchamber at 30  C with a 12-h photoperiod. Vertical bars indicatestandard errors of the mean values. Cuscuta  seed germination and host detection  273   2005 European Weed Research Society  Weed Research  2005  45,  270–278  germination while seeds are in the soil (Hallett &Bewley, 2002). The germination dynamics of   C. campes-tris  is the consequence of a double mechanism of dormancy: after passing through primary dormancy(after-ripening caused by the impermeability of seedcoat), the seeds later go through an annual cycle of secondary dormancy. Emergence  The emergence of   C. campestris  seedlings from a rangeof depths after seed has been stored for 16 months underlaboratory and field conditions is reported in Fig. 3.Laboratory seed storage for 16 months did not breakprimary dormancy and emergence values below 20%were observed. In contrast, field seed storage caused thebreaking of primary dormancy and emergence rangedbetween 60% and 65% for burial depths between 0 and1 cm. For both laboratory- and field-stored seeds, theoptimal sowing depth was 0.5 cm. Similar results wereobserved for other weeds (Mohler & Galford, 1997). Anexplanation of this phenomenon is probably the betterhydration conditions for shallow buried seeds (0.5 cmdepth) compared with unburied seeds. Emergencestrongly declined with increasing seeding depth, regard-less of seed-storage conditions. At a depth of 4 cm,emergence decreased to one-third, compared with aseeding depth of 0.5 cm. For sowing depths >4 cm, noemergence was observed. It is important to note that thelack of emergence is not because of fatal germination, asintact seeds were exhumed from soil after the experi-ment. This lack of germination and emergence is due tothe scarce oxygen presence and relative diffusion withincreasing soil depths (Benvenuti, 2003). Similar depth-mediated emergence has been observed for other small-seed weeds, confirming a relationship between seedweight and emergence ability (Benvenuti  et al. , 2001).This relationship seems to be linked either to quantity orenergy of seed reserves (Milberg  et al. , 1996), whichplays an important role in  C. campestris  during theautotrophic phase of growth. Depth-mediated emer-gence is essential for  Cuscuta , considering that for thisparasitic weed the most sensitive growth phase ends notafter emergence, but following attachment to a host.Finally, a progressive increase of MET as a function of sowing depth was observed (Fig. 4). The MET increasewas observed for both laboratory- and field-stored seedsand no significant differences between the two storageconditions were found ( P  £  0.05). This confirms that theincreased time required for emergence negatively affectssurvival of deeply buried individuals which need tocontact a host. Parasitisation  The percentage of   C. campestris  seedlings reaching asugarbeet host plant of different phenological stages andweed–crop distances are reported in Fig. 5. No statisti-cal differences in the percentages of   Cuscuta  seedlingsreaching sugarbeet at four- or six- to eight-leaf stagewere observed for any weed–crop distance. An increasein the weed–crop distance reduced the number of  Cuscuta  seedlings reaching sugarbeet plants. More than80% of parasitic seedlings were able to reach a host from2 cm. At the distances of 4 and 6 cm, a slight decrease inthe percentage of seedlings reaching a host wasobserved. In contrast, at a distance of 8 cm, thepercentage of seedlings reaching the host was 5% and0 at greater distances. The critical distance from a hostwas between 6 and 8 cm. Considering that the maximumelongation of   C. campestris  seedling was between 7 and8 cm (data not shown), this value corresponded to 01234567801020304050607080 Field storageLaboratory storage Seeding depth (cm)    E  m  e  r  g  e  n  c  e   (   %   ) Fig. 3  Emergence percentage of   C. campestris  seeds, stored for16 months in the laboratory ( d - d ) and in the field ( s - s ), as afunction of increasing burial depths (0, 0.5, 1, 2, 4, 6 and 8 cm).Vertical bars indicate standard errors of the mean values. 0246802468 Laboratory storageField storage Seeding depth (cm)    M  e  a  n  e  m  e  r  g  e  n  c  e   t   i  m  e   (   d  a  y  s   ) Fig. 4  Mean emergence time (MET) of laboratory- ( d - d ) andfield-stored ( s - s )  C. campestris  seeds as a function of increasingburial depths (0, 0.5, 1, 2, 4, 6 and 8 cm). Vertical bars indicatestandard errors of the mean values. 274  S Benvenuti  et al.   2005 European Weed Research Society  Weed Research  2005  45,  270–278
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