W ack bu ial educes ge mina ion and es ablishmen o he in asi e co dg as... 65
W ack bu ial educes ge mina ion and es ablishmen
o he in asi e co dg ass Spa ina densi lo a
Ahmed M. Abbas1, Al edo E. Rubio-Casal1, Al onso de Ci es1,
En ique Figue oa1, Ja ie J. Nie a2, Jesús M. Cas illo1
1 Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de Se illa, A d. Reina
Me cedes, s/n.-41012 Se illa, España 2Depa amen o de Biología Ambien al y Salud Pública, Uni e sidad de
Huel a, Campus del Ca men, 21071 Huel a, España
Co esponding au ho : Jesús Cas illo ([email p o ec ed])
Academic edi o : J. Jeschke|Recei ed 5 Ma ch 2013|Accep ed 13 Oc obe 2013|Published 17 Ap il 2014
Ci a ion: Abbas AM, Rubio-Casal AE, de Ci es A, Figue oa E, Nie a JJ, Cas illo JM (2014) W ack bu ial educes
ge mina ion and es ablishmen o he in asi e co dg ass Spa ina densi lo a. In: Capde ila-A güelles L, Zille i B (Eds)
P oceedings o 7 h NEOBIOTA con e ence, Pon e ed a, Spain. NeoBio a 21: 65–79. doi: 10.3897/neobio a.21.4963
Abs ac
Ge mina ion and eme gence o halophy es may dec ease signi ican ly by seed bu ial in dead plan ma e-
ial, o w ack, which is common and abundan in idal ma shes. The e ec s o plan deb is (w ack) bu ial
on seed ge mina ion and seedling es ablishmen o Spa ina densi lo a, an in asi e co dg ass, we e s udied
unde g eenhouse condi ions and compa ed wi h ield obse a ions. Fi e w ack bu ial dep hs we e ap-
plied: con ol wi hou w ack, 1 cm (1235 ± 92 g DW w ack m-2), 2 cm (3266 ± 13 g DW m-2), 4 cm
(4213 ± 277 g DW m-2), and 8 cm (6138 ± 227 g DW m-2). Sedimen pH, elec ical conduc i i y, edox
po en ial and empe a u e we e eco ded. Quiescence inc eased wi h w ack load up o ~20% a 8 cm deep.
Ge mina ion dec eased wi h w ack load om 96% o 14%, which could be ela ed wi h anoxic condi ions
unde he deb is since sedimen edox po en ial was as low as -83 ± 7 mV a 8 cm. Ge mina ion pe cen -
age inc eased and quiescen and do man pe cen ages dec eased a highe daily sedimen empe a u es and
wi h highe daily empe a u e luc ua ions, condi ions ha we e eco ded wi hou o unde low loads o
w ack. Spa ina densi lo a did no show p ima y do mancy, bu i s seeds en e ed in o a non-deep physi-
ological do mancy below 1 cm deep in plan deb is. The es ablishmen o S. densi lo a seedlings was also
g ea ly educed by w ack bu ial since only 6 seedlings (11 ± 5 % o ge mina ed seeds) eme ged abo e plan
deb is om 1 cm and all seedlings died om deepe han 1 cm. S. densi lo a seedling de elopmen was
also educed by w ack bu ial. The in e se ela ionship be ween ge mina ion and eme gence o S. densi lo a
wi h w ack bu ial eco ded in ou s udy is use ul o p edic i s in asion dynamics and o plan he manage-
men o in aded ma shes.
Copy igh Ahmed Abbas e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC
BY 4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
NeoBio a 21: 65–79 (2014)
doi: 10.3897/neobio a.21.4963
www.penso .ne /jou nals/neobio a
RESEARCH ARTICLE
Ad ancing esea ch on alien species and biological in asions
A pee - e iewed open-access jou nal
NeoBio a
Ahmed Abbas e al. / NeoBio a 21: 65–79 (2014)
66
Keywo ds
Coas al ma shes, physiological do mancy, plan deb is, seedling, Spa ina densi lo a
In oduc ion
Mos sal ma sh en i onmen s a e unp edic able o plan s, because o he occu ence
o bo h salini y and looding s ess (Can e o e al. 1998). When condi ions o seed
ge mina ion a e no a o able, unge mina ed seeds o halophy es o en emain unde
en o ced do mancy in he soil and se e as a ansien o pe sis en seed bank (Unga
1995). Thus, seed banks a e pa icula ly impo an in main aining popula ions o hal-
ophy es in saline ma shes (Co e and Van Auken 2006). Sal ma shes a e among he
mos hea ily in aded sys ems in he wo ld (G osholz 2002). The abili y o in oduced
species o bank seeds can con ibu e o in asion success, since seeds can pe sis while
wai ing o a o able condi ions. This abili y is especially use ul in en i onmen s whe e
oppo uni ies o seed ge mina ion a e in equen o unp edic able such as sal ma shes
(Pa ke e al. 1989). Managemen o in asi e species in sal ma shes can be challenging
because moni o ing and con ol mus con inue o a leas as long as hei seeds pe sis
in he seed bank (Pane a and Timmins 2004).
Ra s o dead plan ma e ial, o w ack, a e common and abundan in idal ma sh-
es. Due o high p ima y p oduc i i y a es and low consump ion a es by he bi o-
ous, halophy es, such as eelg asses and co dg asses, p oduce high quan i ies o w ack,
especially in dye-back a eas. This w ack is added up o ha anspo ed by i e s o
sal ma shes in es ua ies. Then, idal c eeks p o ide co ido s o w ack anspo in o
sal ma shes whe e i can dis u b plan communi ies (Reidenbaugh and Ban a 1980;
Be ness and Ellison 1987; Valiela and Rie sma 1995; Tolley and Ch is ian 1999;
B ewe e al. 1998; Minchin on 2002). As pa o he na u al dis u bance egime, de-
posi ed w ack ma s o en c ea e ba e spo s by killing no only he su ace ege a ion,
bu also he below-g ound biomass (Ha man 1988). Seeds o di e en sal ma sh
plan s can ge mina e in he spaces opened by w ack accumula ion, playing a key ole
in plan dis ibu ion (Ha man e al. 1983; Pennings and Richa ds 1998), bu ge mi-
na ion and eme gence may dec ease signi ican ly by seed bu ial in plan deb is. Bu -
ied seeds a e ec ui ed only when hey a e b ough back o he su ace by dis u bances
(Facelli and Picke 1991). Howe e , hese e ec s a e poo ly unde s ood since he e
is a gene al lack o esea ch ha examines he e ec s o w ack bu ial in sal ma shes.
The aus al co dg ass, Spa ina densi lo a B ongn. (Poaceae) is in ading sal ma shes
in sou he n Eu ope, No hwes A ica and he Wes Coas o No h Ame ica (Bo olus
2006). In in aded ma shes, S. densi lo a de elops e y dense popula ions whe e la ge
amoun s o dead ma e a e deposi ed (Nie a e al. 2001; Cas illo e al. 2008). Mo eo-
e , S. densi lo a seeds loa so hey a e dispe sed by cu en s and ides oge he wi h
plan deb is (Howa d and Sy sma 2013). One o he keys o he success o S. densi lo a
in asion is i s abili y o p oduce la ge quan i ies o iable seeds (Nie a e al. 2001a,b).
W ack bu ial educes ge mina ion and es ablishmen o he in asi e co dg as... 67
Spa ina densi lo a p oduces many seedlings in in e idal mud la s bu i s ec ui men
is e y low in ma sh zones whe e w ack is accumula ed.
While co dg asses (Spa ina genus) a e one o he mos abundan and equen hal-
ophy es in sal ma shes, he e ha e no been any s udies ha examined how hei seeds
and seedlings espond o w ack bu ial. We examined unde con olled g eenhouse con-
di ions he impac o i e w ack bu ial dep hs (0, 1, 2, 4 and 8 cm) on ge mina ion,
seed iabili y and seedling es ablishmen o S. densi lo a. Main abio ic ac o s condi-
ioning seed ge mina ion and seedling es ablishmen in sal ma shes (sedimen pH,
elec ical conduc i i y, edox po en ial and empe a u e) we e also eco ded o e e y
w ack ea men . We hypo hesized ha S. densi lo a ge mina ion and es ablishmen
would be educed by w ack bu ial due o anoxia and/o low empe a u e luc ua ions.
Me hods
Seed and w ack collec ion
Spa ina densi lo a seeds we e collec ed in Augus 2009 om mul iple ma u e indi-
iduals chosen a andom om a popula ion g owing a he pe iphe y o an acc e ing,
well-d ained in e idal lagoon a Odiel Ma shes (Sou hwes Ibe ian Peninsula; 37°08'
- 37°20'N, 6°45' - 7°02'W; Fig. 1); see Cas ellanos e al. (1994) o a desc ip ion o
he si e. Seeds we e s o ed a 5 °C, unde d y condi ions and in da kness a e ha es
un il he beginning o he expe imen . W ack was collec ed om he mean highe high
wa e (MHHW) om he same ma sh and i consis ed o dead culms and lea es o
di e en plan species (~50% o he deb is was dead S. densi lo a) oge he wi h pieces
o shells (diame e < 1.0 cm). Collec ed w ack was checked o he p esence o S.
densi lo a seeds and none was ound, p obably because seeds a e dispe sed be o e dead
spiked shoo s a e anspo ed by ides and cu en s.
W ack ea men s
A g eenhouse expe imen was conduc ed om Decembe 2009 o Feb ua y 2010,
since mos S. densi lo a seeds ge mina e du ing win e , o es he e ec s o w ack bu ial
on seed ge mina ion, and eme gence and g ow h o seedlings o S. densi lo a. The mean
disseminule size o S. densi lo a was 9.67 ± 0.15 mm by 1.47 ± 0.02 mm (n = 50) and
i s mean weigh was 3.0 ± 0.1 mg ( ange: 1.3-4.2 mg). Mean Spa ina ca yopsis size
was 4.68 ± 0.08 mm by 0.98 ± 0.02 mm (n = 50) and i s weigh was 2.0 ± 0.1 mg
( ange: 1.3–3.0 mg). Fou eplica es o 25 seeds we e sown a 1 cm dep h in clean sand
in plas ic con aine s measu ing 18 cm wid h, 22 cm leng h and 11 cm heigh (con-
aining ~1.6 kg o clean sand). The sand was collec ed om he same ma sh whe e he
seeds we e ob ained and i was sie ed h ough 0.5-mm mesh size illing o elimina e
p e-exis ing seeds and o he plan ma e ial. A con ol ea men was se up wi hou any
Ahmed Abbas e al. / NeoBio a 21: 65–79 (2014)
68
seeds added in o de o es whe he he sand we used con ained seeds. Fi e w ack bu -
ial ea men s we e conduc ed: con ol (no w ack was added abo e he sand su ace),
1 cm (1235 ± 92 g DW w ack m-2), 2 cm (3266 ± 13 g DW m-2), 4 cm (4213 ± 277 g
DW m-2), and 8 cm (6138 ± 227 g DW m-2) o w ack bu ial dep h. These ea men s
we e decided ollowing ou ield obse a ions in Odiel Ma shes (Sou hwes Ibe ian
Figu e 1. Loca ion o he Odiel Ma shes on he A lan ic coas o Sou hwes Ibe ian Peninsula (37°08'-
37°20'N, 6°45'-7°02'W), and he sampling poin whe e Spa ina densi lo a seeds we e collec ed (1).
W ack bu ial educes ge mina ion and es ablishmen o he in asi e co dg as... 69
Peninsula) whe e S. densi lo a is e y abundan (Nie a e al. 2001a). Each con aine
had 25 seeds a one dep h, so he e we e i e ea men s wi h ou con aine s ( epli-
ca es) pe dep h (n = 4 pe ea men ; n = 20, including all ea men s oge he ). Con-
aine s we e i iga ed gen ly once a day wi h wa e o ensu e he mois u e o he soil
emained wi hin 70% o i s wa e -holding capaci y. F esh wa e (< 0.5 psu) was used
o a oid salini y e ec s on ge mina ion since we wan ed jus o eco d seed esponses
o bu ial and a oid high salini y e ec s. Spa ina densi lo a is a acul a i e halophy e
ha can ge mina e, es ablish and de elop in eshwa e condi ions (Nie a e al. 2001b;
Cas illo e al. 2005). The con aine s had small holes on he bo om o allow d ainage,
bu hese we e co e ed wi h s ips o clo h o p e en he loss o sand.
Sedimen edox po en ial, elec ical conduc i i y and pH we e eco ded a he end
o he expe imen in Feb ua y 2010 a 1 cm dep h in he sand. pH was eco ded in he
labo a o y a e adding dis illed wa e o he soil (1:1, soil: dis illed wa e , / ) (pH/ edox
C ison wi h he elec ode M-506). Soil salini y was measu ed as elec ical conduc i i y
(conduc i i y me e , C ison-522) a e pH (1:2, soil: dis illed wa e , / )). Redox po en-
ial was de e mined wi h a po able me e and elec ode sys em in he g eenhouse (C ison
pH/mV p-506). Mean, maximum and minimum daily sedimen empe a u e we e e-
co ded a 0, 1, 2, 4 and 8 cm dep hs using an elec onic he mome e (SA880SSX, Ge -
many) eco ding om 8 a.m. o 8 p.m. e e y 4 hou s o 3 days (n = 3). A e age daily ai
empe a u e du ing he expe imen was 18.5 ± 0.5 °C, a ying be ween a mean low em-
pe a u es o 10.4 ± 1.6 °C and a mean maximum empe a u e o 34 ± 3.5 °C. Mean daily
ai ela i e humidi y was 74.5 ± 2.6%, a ying be ween 32.5 ± 5.5% and 92.5 ± 1.1%.
Eme ged seedlings om benea h he w ack we e coun ed e e y 24 h (Cui e al.
2007). The expe imen con inued un il no addi ional eme gence was obse ed du ing
10 days. A he end o he expe imen , he w ack and he sand we e ca e ully emo ed
and hose seedlings ha died be o e aising he w ack su ace we e coun ed and unge -
mina ed seeds we e collec ed. A he end o he expe imen , shoo and oo leng h we e
measu ed on all seedlings and he numbe o oo s coun ed. Ge mina ion pe cen age
was calcula ed as he numbe o ge mina ed seeds by he o al numbe o seeds pe
ea men . Seedling eme gence pe cen age was calcula ed as he numbe o seedlings
aising abo e he w ack su ace by he numbe o ge mina ed seeds pe ea men .
Seedling ae ial g ow h a e was eco ded as he di e ence in eme ged seedling heigh
be ween wo consecu i e measu es (in wo days pe iod) di ided by he numbe o days.
Unge mina ed seeds we e ans e ed o 0.5-cm dep h in sand and ge mina ion and
eme gence pe cen ages we e eco ded un il no mo e seeds ge mina ed o 10 days. Ge -
mina ed seeds we e conside ed o be in a quiescen s a e, which is di e en han ue
seed do mancy and occu s when a seed ails o ge mina e because ex e nal en i onmen al
condi ions a e no app op ia e (Baskin and Baskin 1985). Then, unge mina ed seeds
we e soaked in wa e a 30 °C o 24 h. Seed coa s we e cu and he emb yo was soaked
in 1% e azolium chlo ide (Pan eac Quimica S.A., Ba celona, Spain) o 24 h a 30 °C.
Pink emb yos we e sco ed as ali e and conside ed o be in do mancy (Baskin and Baskin
2004). Non-colou ed seeds we e conside ed o be dead. Quiescence, do mancy and mo -
ali y pe cen ages we e calcula ed in ela ion o he o al numbe o seeds pe ea men .

Ahmed Abbas e al. / NeoBio a 21: 65–79 (2014)
70
S a is ical analysis
Analyses we e ca ied ou using SPSS 12.0 (SPSS Inc, USA). Da a we e es ed o
homogenei y o a iance and no mali y wi h he B own-Fo sy he es and he Kolmog-
o o -Smi no es , espec i ely (P < 0.05). Plan ai s we e compa ed be ween ea -
men s by one-way analysis o a iance (ANOVA, F- es ). Tukey Hones Signi ican
Di e ence (HSD) es be ween means was calcula ed only i he F- es was signi ican
(P < 0.05). The e ec o w ack load on seedling g ow h a e be ween wo ea men s
was analyzed using a S uden - es . Pea son co ela ion coe icien and linea eg es-
sions we e calcula ed be ween abio ic ac o s, ge mina ion and seedling ai s, and he
w ack load. When a bio ic cha ac e is ic was co ela ed wi h wo o mo e abio ic en-
i onmen al ac o s, mul iple eg ession analysis was ca ied ou o explo e ela i e
weigh s (Ƣ). De ia ions we e calcula ed as s anda d e o s o he mean (SEM).
Resul s
Abio ic en i onmen
Sedimen pH inc eased wi h he w ack bu ial load om 6.2 ± 0.5 in he con ol ea -
men o 7.7 ± 0.1 unde 8 cm ( = 0.96, P < 0.01, n = 20). Elec ical conduc i i y changed
om 0.11 ± 0.00 o 0.23 ± 0.00 mS cm-1. Sedimen edox po en ial a ied be ween -83
± 7 mV unde 8 cm o w ack and +255 ± 5 mV wi hou w ack bu ial (Table 1). Mean
daily sedimen empe a u e dec eased a highe dep hs ( = -0.61, P < 0.05, n = 20), bu
wi hou showing signi ican di e ences be ween ea men s, a ying be ween 12.3 ± 1.0
°C o he con ol ea men and 10.1 ± 0.8 °C a 4 and 8 cm (ANOVA, F = 0.39, P >
0.05). Maximum daily sedimen empe a u e changed be ween +25.0 ± 0.8 °C o he
con ol ea men and +21.7 ± 0.3 °C a 1 cm (ANOVA, F = 2.02, P > 0.05), dec easing
also when w ack bu ial dep h inc eased ( = -0.36, P > 0.05, n = 20). Minimum daily
empe a u e a ied be ween +10.4 ± 0.4 °C a 8 cm and +7.9 ± 1.0 °C o he con ol
ea men (ANOVA, F = 0.86, P > 0.05), inc easing wi h dep h ( = 0.81, P < 0.0001,
n = 20). Daily a ia ion be ween maximum and minimum empe a u es dec eased a
highe dep hs ( = -0.62, P < 0.05, n = 20), a ying be ween 17.7 ± 1.4 °C o he con ol
ea men and 12.4 ± 0.3 °C a 8 cm (ANOVA, F = 2.46, P < 0.05) (Table 1).
Ge mina ion and es ablishmen
The ime o i s eme gence o S. densi lo a was 23 days a e he s a o he expe i-
men a con ol ea men and 37 days a 1 cm w ack dep h. Ge mina ion dec eased
when w ack load inc eased ( = -0.84, P < 0.0001, n = 20), showing he highes alue
wi hou w ack (96 ± 4%) and dec easing signi ican ly unde 8 cm deep (ANOVA, P
< 0.05) (Fig. 2).
W ack bu ial educes ge mina ion and es ablishmen o he in asi e co dg as... 71
Table 1. W ack load (g m-2), sedimen pH, edox po en ial (mV), elec ical conduc i i y (mS cm-1 ), daily mean, maximum and minimum sedimen empe a u e
(°C), and he di e ence be ween maximum and minimum empe a u e (°) (n = 3) o i e w ack bu ial dep hs. Di e en le e s indica e signi ican di e ences be ween
ea men s (ANOVA, P < 0.05) (n = 5).
W ack dep h
(cm)
W ack load
(gm-2)pH Redox po en ial
(mV)
Conduc i i y
(mS cm-1 )Daily sedimen empe a u e (°C)
Mean Maximum Minimum Max-Min
0 0 ± 0a6.2 ± 0.5a+255 ± 5a0.23 ± 0.00a12.3 ± 1.0a25.0 ± 0.8a7.9 ± 1.0a17.7 ± 0.2a
1 1235 ± 92b6.9 ± 0.3b+247 ± 2a0.20 ± 0.02a10.4 ± 0.9a21.7 ± 0.3a9.4 ± 0.5a13.0 ± 0.3b
2 3266 ± 13c7.1 ± 0.5b+229 ± 3b0.16 ± 0.00b10.2 ± 0.9a22.5 ± 0.1a9.7 ± 0.5a13.1 ± 0.0b
4 4213 ± 277d7.3 ± 0.1c+157 ± 11c0.13 ± 0.01c10.1 ± 0.8a22.7 ± 0.8a10.1 ± 0.4a12.7 ± 0.1b
8 6138 ± 227e7.7 ± 0.1d-83 ± 7d0.11 ± 0.00d10.1 ± 0.8a22.5 ± 0.0a10.4 ± 0.4a12.4 ± 0.3b
Ahmed Abbas e al. / NeoBio a 21: 65–79 (2014)
72
Ge mina ion pe cen age dec eased a lowe edox po en ials ( = 0.65, P < 0.005,
n = 20; Ƣ = -0.599), a ying om 96 ± 4% a he con ol ea men wi h sedimen
edox po en ial +255 ± 5 mV o less han 15% unde 8 cm deep wi h nega i e edox
po en ial -83 ± 7 mV. In addi ion, ge mina ion pe cen age inc eased a lowe mini-
mum daily sedimen empe a u e ( = -0.95, P < 0.0001, n = 20; Ƣ = -3.669) (Fig. 2).
Quiescence and do mancy pe cen ages inc eased wi h w ack load ( = 0.67, P <
0.05, n = 20; = 0.70, P < 0.001, n = 20, espec i ely). Quiescen pe cen age inc eased
a highe minimum daily sedimen empe a u e ( = 0.57, P < 0.05, n = 20; Ƣ = 3.265)
and a lowe edox po en ials ( = -0.56, P < 0.01, n = 20; Ƣ = 0.495). No do man
seeds we e eco ded o he con ol ea men whe e seed mo ali y was he lowes
(4%; 4 seeds). Do mancy and mo ali y inc eased o ~50% o he unge mina ed seeds
unde w ack (Fig. 2). Do man seed pe cen age and seed mo ali y inc eased mainly a
highe daily minimum sedimen empe a u e ( = 0.70, P < 0.001, n = 20; = 0.81, P
< 0.0001, n = 20, espec i ely).
No seedling eme ged om deepe han 1 cm dep h since e e y seedling died be-
o e eme ging abo e he w ack su ace. Minimum seedling mo ali y was eco ded o
he con ol ea men (7 ± 3% o ge mina ed seeds) (ANOVA, P < 0.05). The highes
seedling eme gence pe cen age occu ed wi hou w ack (93 ± 3%) and only 6 seedlings
eme ged om unde 1 cm o w ack (11 ± 5%) (Table 2).
Seedlings in he con ol ea men we e much alle and had longe and mo e oo s
han hose g owing om unde 1 cm o w ack and also han hose seedlings dying un-
de he w ack a 2, 4 and 8 cm (Fig. 3). These esul s also showed a highe ae ial g ow h
a e o seedlings a he con ol ea men (0.11 ± 0.01 cm day-1) han hose g owing
om unde 1 cm o w ack (0.02 ± 0.01 cm day-1) ( - es ; P < 0.05).
Figu e 2. Ge mina ion, quiescence, do mancy and mo ali y o Spa ina densi lo a seeds o i e w ack
bu ial dep hs.
W ack bu ial educes ge mina ion and es ablishmen o he in asi e co dg as... 73
Discussion
This s udy shows ha ge mina ion and es ablishmen o S. densi lo a, an in asi e
co dg ass in Eu ope, No h Ame ica and No h A ica, is g ea ly limi ed by w ack bu ial.
Sedimen pH inc eased wi h w ack bu ial load, which may be ela ed o he p esence
o shells in he deb is ha would add ca bona e o he sedimen . Howe e , S.densi lo a
ge mina ion would no be al e ed wi hin he na ow eco ded pH ange (6.2–7.7) (Cu ado
e al. 2010). Simila ly, elec ical conduc i i y a ied wi hin a ange (0.11–0.23 mS cm-1)
ha would no in luence S. densi lo a ge mina ion signi ican ly (Cas illo e al. 2005). In
con as , sedimen edox po en ial unde he deb is may ha e a ec ed ge mina ion unde 8
cm o w ack since i was as low as -83 ± 7 mV, and ge mina ion dec eased and quiescence
inc eased a low edox po en ials as i has been desc ibed p e iously in anae obic en i on-
Table 2. Eme gence and mo ali y a es o ge mina ed seeds o i e w ack bu ial dep hs. Di e en le -
e s indica e signi ican di e ences be ween w ack bu ial dep hs o he same ai (ANOVA, P < 0.05).
W ack dep h (cm) Eme gence (%) Mo ali y (%)
0 93 ± 3a7 ± 3a
1 11 ± 5b89 ± 5b
2 0 ± 0c100 ± 0c
4 0 ± 0c100 ± 0c
8 0 ± 0c100 ± 0c
Figu e 3. Shoo and oo leng hs (cm) and oo numbe o Spa ina densi lo a seedlings o i e w ack bu ial
dep hs (seedlings om deepe han 1 cm we e ound dead and did no eme ge abo e he w ack su ace).
Di e en le e s indica e signi ican di e ences be ween w ack dep hs o he same ai (ANOVA, P < 0.05).