Revista de la Facultad de Ciencias
Agrarias. Universidad Nacional de Cuyo. En prensa. ISSN (en línea) 1853-8665.
Original article
The Native Dryland PGPR ‘Pseudomonas
42P4’ Promotes Adventitious Rooting in Woody Cuttings of Vitis spp.
La
PGPR nativa de zonas áridas ‘Pseudomonas 42P4’ promueve la fomación de
raíces adventicias en estacas leñosas de Vitis spp.
Maria Gabriela
Gordillo1,
Fanny Colombo3,
1Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias.
Instituto de Biología Agrícola de Mendoza (IBAM-CONICET). Almirante Brown 500.
Chacras de Coria. M5528AHB. Mendoza. Argentina.
2Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias.
Almirante Brown 500. Chacras de Coria. M5528AHB. Mendoza. Argentina.
3Trapiche Winery. Calle Nueva Mayorga s/n. Maipú. Mendoza.
Argentina. CPA: M5513
*cgonzalez@fca.uncu.edu.ar
Abstract
This study
evaluated the effect of two native PGPR strains from an arid region (Mendoza,
Argentina) on the rooting of woody cuttings of Vitis spp. These strains
are known for their growth-promoting capacity, including auxin production.
Dormant V. vinifera cv. Malbec cuttings were grafted onto four
rootstocks - 1103 Paulsen, 110 Richter, 101-14 MGt and SO4. Then, basal ends of
these grafted cuttings and own rooted controls were incubated for 12 h in
solutions of (1) Pseudomonas 42P4 at 107 CFU mL-1,
(2) Enterobacter 64S1 at 107 CFU mL-1,
(3) autoclaved LB medium, (4) water, and (5) a quick-dip immersion of
Indole-3-butyric acid (IBA). After treatment, the cuttings were placed in a
forcing chamber at 28°C and relative humidity ~100% for 21 days. Rooting
parameters and scion-rootstock union percentages were recorded. Pseudomonas 42P4
significantly promoted rooting in Malbec own-rooted cuttings. However, Enterobacter
64S1 had negative or null effects. Furthermore, Pseudomonas 42P4
enhanced rooting in Malbec grafted onto 1103 Paulsen, but not on 101-14 MGt,
110 Richter or SO4. This strain also improved graft union success on SO4, but
did not affect the other rootstocks. These results suggest that a dryland
native strain such as Pseudomonas 42P4 could sustainably enhance the quality of
both own-rooted and grafted grapevine plants in commercial nurseries.
Keywords: PGPR, grapevine
rootstock, Malbec, rooting, graft union, nursery
Resumen
El objetivo de este
estudio fue evaluar el efecto de dos cepas PGPR nativas de zonas áridas
(Mendoza, Argentina) con capacidad promotora del crecimiento (que producen
auxinas) sobre el enraizamiento de estacas leñosas de Vitis spp. La base
de las estacas de Vitis vinifera cv. Malbec, tanto francas como
injertadas sobre cuatro portainjertos:1103 Paulsen, 110 Richter, 101-14 MGt y
SO4, se incubaron durante 12 h en soluciones de 1) Pseudomonas 42P4 y 2)
Enterobacter 64S1 107 UFC mL-1,
3) medio LB autoclavado, 4) agua y 5) una inmersión rápida de ácido
indol-3-butírico (IBA). Posteriormente, las estacas se colocaron en una cámara
de forzadura a 28°C y humedad relativa ~100% durante 21 d. Se midieron
diferentes parámetros de enraizamiento y el porcentaje de unión del injerto.
Los resultados mostraron que Pseudomonas 42P4, pero no Enterobacter 64S1
promovió el enraizamiento, de forma similar a IBA en estacas de Malbec. La cepa
Pseudomonas 42P4 también promovió el enraizamiento de estacas injertadas
sobre 1103 Paulsen, pero no sobre 101-14, 110 Richter y SO4. Además, Pseudomonas
promovió la unión del injerto en SO4, pero no en 110 Richter, 1103 Paulsen
y 101-14. Estos resultados sugieren que una cepa nativa de zonas áridas podría
utilizarse como una herramienta sustentable para mejorar la calidad de plantas
francas e injertadas de vid.
Palabras clave: PGPR, portainjertos
de vid, Malbec, enraizamiento, unión del injerto, vivero
Originales: Recepción: 16/04/2025
- Aceptación: 06/07/2025
Introduction
A significant portion
of worldwide agricultural land (45%) is dryland. Climate change (CC) threatens
agroecosystems, causing detrimental effects on crop productivity (Berdugo et al., 2020; Burrel et al., 2020).
Viticulture covers 7.3 million hectares of the world, with over 60% of grapes
produced in drylands (OIV 2023; Flexas et al.,
2010). Argentina ranks seventh, with the largest cultivated vineyard area
globally. This country mainly develops irrigated viticulture in drylands (OIV 2023, INV 2023).
Grapevines are
clonally propagated through one-year woody cuttings. Grafted or ungrafted woody
cuttings are forced under high relative humidity (RH) and ± 27°C to stimulate
adventitious root formation and scion-rootstock healing (in grafted plants).
External factors (temperature, humidity, substrate aeration) and internal
factors (carbohydrate reserves, hormones, and genotype) influence these
processes (Hartmann et al., 2014).
Rootstocks exhibit
resistance to pathogens and diseases, and confer tolerance to abiotic stresses
(Hartmann et al., 2014; Ollat et al.,
2016, Keller, 2020, D’Innocenzo et al., 2024). V. vinifera cultivars
are generally grafted onto American Vitis spp. hybrid
rootstocks due to their resistance to phylloxera (Mudge et
al., 2009). Grafting is essential in most European wine-growing
regions. However, outside Europe, vineyards can be planted with own-rooted V.
vinifera plants.
One main problem in
grapevine propagation is the differential rooting capacity of rootstocks.
Rootstocks 110 Richter (110 R) and Selection Oppenheim 4 (SO4) are recalcitrant
to form adventitious roots. This differs from other widely used grapevine
rootstocks like 1103 Paulsen (1103 P) and 101-14 Millardet et de Grasset
(101-14 MGt), which induce more root development (Keller, 2020). Low rooting
capacity causes significant economic losses to nurseries. Additionally,
scion-rootstock interaction plays a crucial role in rooting. The scion may
affect root dry weight per woody cutting (Tandonnet et
al., 2009), as the scion-rootstock union simultaneously occurs with
rooting, depending on cutting reserves.
Auxins are the main phytohormones in adventitious root
production of woody cuttings (Burnoni et al.,
2022), though their effect varies among genotypes. For example,
indole-3-butyric acid (IBA), a primary synthetic auxin used for grapevine
rooting (Machado et al., 2005), promotes
rooting in woody cutting of 110 R, SO4 and 101-14 MGt (Gordillo
et al., 2022; Satisha et al., 2008). In contrast, IBA may
promote (Satisha et al., 2008; Daskalakis et
al., 2018) or not (Boeno et al., 2023;
Gordillo et al., 2022) rooting of 1103 P woody cuttings. However,
synthetic agrochemicals are progressively being excluded as organic and
agroecological agriculture gains attention (Centeno et
al., 2008).
Plant Growth-Promoting
Rhizobacteria (PGPR) are plant symbiotic bacteria that colonize the rhizosphere
and produce indole acetic acid (IAA)-type auxins (Glick et
al., 2012; Pantoja Guerra et al., 2023). Some PGPR strains
promote rooting of difficult-to-root rootstocks, while others have no effect (Isçi et al., 2019; Toffanin et al., 2016).
Our group isolated and characterized two PGPR strains from Mendoza´s arid soils
of Argentina. Pseudomonas 42P4 (42P4) and Enterobacter 64S1
(64S1) produce IAA (Pérez-Rodríguez et al., 2020a)
and promote seedling growth of Solanum lycopersicum (Pérez-Rodríguez et al., 2022) and Capsicum
annuum seedlings (Lobato Ureche et al., 2021),
alleviate saline stress in tomato (Pérez-Rodríguez et
al., 2022), and increase drought tolerance in Arabidopsis thaliana plants
(Jofré et al., 2024). Furthermore, Pseudomonas
42P4 enhances tomato growth, yield and fruit quality under field conditions
(Pérez-Rodríguez et al., 2020b). Both strains
also exhibit biocontrol activity, inhibiting tomato and pepper crop diseases
(unpublished data). Native strains easily adapt to edaphic conditions, resist
local environmental stresses, and are more successful when inoculated into the
plant rhizosphere.
PGPR improve
rooting and survival of young plants of various species. However, few studies
report PGPR´s effect on woody plant production or grapevine woody cuttings,
specifically (Bartolini et al., 2017; Köse et
al., 2003, 2005; Tofanin et al., 2016). Currently, exploring
sustainable tools is imperative, especially given global warming threats,
particularly challenging in drylands.
Therefore, this
work evaluated the effect of two native PGPR strains from Mendoza’s arid soils,
Pseudomonas 42P4 and Enterobacter 64S1, on adventitious root
production of the Argentinean emblematic cultivar: Malbec, and the four most
widespread rootstocks in global viticulture and Argentine arid areas: SO4, 110
R, 1103 P, and 101-14 MGt (Riaz et al., 2019).
These strains, adapted to arid soils, could constitute a sustainable
alternative for synthetic agrochemicals in grapevine propagation.
Materials
and Methods
Bacterial
Culture
Pseudomonas 42P4 (42P4) and Enterobacter
64S1 (64S1) were collected, isolated and characterized by the Plant
Physiology and Microbiology Group (IBAM- FCA, CONICET-UNCuyo, Mendoza,
Argentina). The partial 16S rRNA sequence of both strains was deposited in
GenBank under accession numbers MT045993.1 and MT047267, respectively (Pérez-Rodríguez et al., 2020a). Inocula were
prepared in 1 L Erlenmeyer Flask with 400 mL of Luria Broth (LB) culture medium
(10 g Peptone, 5 g Yeast Extract, 5 g NaCl in 1 L of bidistilled H2O).
Bacteria were cultured in an orbital shaker at 120 rpm and 32°C for 24 h.
Strain concentration was estimated by optical density at 540 nm in a
spectrophotometer according to Pérez-Rodriguez et al.
(2020a). From these cultures, a dilution to 107 colony-forming units
(CFU) mL–1 was prepared for each
strain in PBS (phosphate buffer saline).
Plant
Material, Inoculation and Rooting Conditions
Two experiments
were conducted in the grapevine nursery of Grupo Peñaflor S.A. (Trapiche
Winery), located in Santa Rosa, Mendoza, Argentina (33°15‘39.4” S 68°07’48.9”
W). The first experiment was conducted during the 2020 season
(September-October) and the second, in 2021. Cuttings (length: 40 cm and
diameter: 9 mm) were collected in winter and stored at 4°C until experiment
initiation.
In the first
experiment, we tested different doses of Pseudomonas 42P4 and Enterobacter
64S1 on the rooting capacity of V. vinifera cv. Malbec woody
cuttings. Before forcing, we applied five treatments: (1) 30-second quick
immersion in 1000 ppm IBA solution (Gordillo et al.
2022); or 12-hour incubation in solutions of: (2) Pseudomonas 42P4
at 107 CFU mL-1,
(3) Enterobacter 64S1 at 107 CFU
mL-1, (4)
autoclaved LB, and (5) tap water (control treatment) on the base (1.5 cm) of 50
woody cuttings per treatment.
In a second
experiment, we evaluated Pseudomonas 42P4 on the rooting of Malbec
cuttings (with 2 buds and approximately 5 cm long) grafted onto cuttings of
101-14 MGt (V. riparia × V. rupestris), SO4 (V. berlandieri × V.
riparia), 1103 Paulsen, and 110 Richter (V. berlandieri × V. rupestris),
with 5 buds (approximately 35 cm long and 9 mm in diameter). We used an Omega grafting
machine (Fornasier Cesare & C., Italy). Each grafted cutting was 40 cm
long. The grafting zone was covered with a commercial initiation wax (Guerowax
Crecimiento 78, Guerola Industries, Spain) to prevent dehydration and diseases
during forcing. Before forcing we applied the following treatments: (1) a
30-second quick immersion at 750 ppm IBA (Gordillo et
al. 2022), and 12-hour incubations with: (2) Pseudomonas 42P4 at
107 CFU mL-1,
(3) autoclaved LB, and (4) tap water (control) on the base (1.5 cm) of 50
grafted cuttings per treatment.
Subsequently, in
both experiments, cuttings were horizontally placed in plastic boxes with peat
(KEKKILÄ professional, https://www.kekkilaprofessional.com/). Then, cuttings
were forced and maintained for 21 days at 28°C and 100% RH.
The experimental
design was a completely randomized design, considering each cutting as an
experimental unit.
Morphological
Parameters
Treatment effect
was evaluated by callus and rooting percentages, followed by root number and
biomass (g) per cutting. Rooting percentage is the percentage of cuttings that
produced at least one root. Callus percentage was visually determined as the
proportion of the cutting base occupied by callus (0-25, 25-50, 50-75, and
75-100%). Root biomass was determined as dry weight (DW) of all adventitious
roots from a cutting, oven-dried at 60°C to constant weight. In grafted
cuttings, the percentage of scion-rootstock union was visually determined as
well (0-25, 25-50, 50-75, and 75-100%).
Statistical
Analysis
Statistical
analyses were performed using the InfoStat P 2020v software (Di Rienzo et al., 2020). Rooting, callus and
graft union percentages, along with root number and biomass, were analysed with
Generalized Linear Models. A binomial distribution was used for the first three
parameters and the Poisson distribution for the fourth. Root biomass was first
tested for ANOVA assumptions (using Shapiro-Wilks test for normality and Levene
test for homoscedasticity). Different letters indicate significant differences
among treatments according to the post-hoc test DGC (α = 0.05). Data
visualization was conducted in R (R Core Team, 2024)
and the ggplot2 package (Wickham, 2016).
Results
Own-rooted
Malbec cuttings
Basal callus
percentage on Malbec was similar among treatments (>75%, p > 0.05) (figure 1A). Pseudomonas 42P4 and IBA 1000 ppm increased
rooting percentage (>80%), compared to autoclaved LB and water treatments
(70%). However, Enterobacter 64S1 decreased rooting percentage (25%)
compared to water (figure 1B). The number of roots per
cutting was similar in cuttings incubated with 42P4 and IBA (~ 6 roots per
cutting), and higher than in the remaining treatments (~ 4 roots per cutting) (figure 1C). Root biomass per cutting was 30% higher in cuttings
incubated in IBA than in 42P4. The remaining treatments yielded lower biomass (figure 1D). Pseudomonas 42P4 promoted three of the four
rooting parameters compared to the water control: rooting percentage, number of
roots per cutting, and root biomass. In contrast, the native Enterobacter 64S1
strain did not promote any evaluated parameter.
Based on these results, we assessed the ability of the native Pseudomonas
42P4 strain (but not Enterobacter) to promote rooting and graft
union of Malbec cuttings grafted onto four grapevine rootstocks.
A)
Callus percentage, B) Rooting percentage, C) Number of roots per cutting, and
D) Root biomass per cutting.
Values
correspond to adjusted means ± SEM (n=50). Data were analysed with General or
Generalized Mixed Linear Models. Different letters indicate significant
differences among treatments according to the post-hoc test DGC (α = 0.05).
42P4:
Pseudomonas 42P4, 64S1: Enterobacter 64S1, IBA: Indole-3-butyric
acid, LB: Luria Broth culture medium, water: tap water.
A)
Porcentaje de callo, B) Porcentaje de enraizamiento, C) Número de raíces por
estaca y D) Biomasa de raíces por estaca.
Los
valores corresponden a las medias ajustadas ± EE (n=50). Los datos fueron
analizados mediante Modelos Lineales Mixtos Generales o Generalizados. Letras
diferentes indican diferencias significativas entre tratamientos según el test
post-hoc DGC (α = 0,05).
42P4: Pseudomonas
42P4, 64S1: Enterobacter 64S1, IBA: ácido indol-3-butírico, LB:
medio de cultivo Luria Broth, water: agua de red.
Figure
1. Own-rooted Malbec cuttings.
Figura 1. Estacas
de Malbec a pie franco.
Grafted
Cuttings
1103 Paulsen
The graft union percentage (~75%) between Malbec and 1103 P
rootstock, and callus percentage (~100%, p > 0.05, data not shown) were
unaffected by treatments (figure 2A). Rooting percentage was
higher (~90%) in cuttings incubated with Pseudomonas 42P4 (figure
2B) than in other treatments (~75%). The number of roots per cutting was
duplicated in 42P4 and IBA treatments than in the controls (water and LB) (figure 2C). However, root biomass was 10% higher in cuttings
incubated with 42P4 and IBA compared to the other treatments (figure
2D). Pseudomonas 42P4 promoted a 15% increase in rooting percentage
compared to IBA, while matching root number and biomass results to this hormone
treatment.
A)
Scion-rootstock union percentage, B) Rooting percentage, C) Number of roots per
cutting, and D) Root biomass per cutting. Values correspond to adjusted means ±
SEM (n=50). Data were analysed with General or Generalized Linear Models.
Different letters indicate significant differences between treatments according
to the post-hoc test DGC (α = 0.05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter
64S1, IBA: Indole-3-butyric acid, LB: Luria Broth culture medium, water:
tap water.
A) Porcentaje de
unión entre la púa y el portainjerto, B) Porcentaje de enraizamiento, C) Número
de raíces por estaca y D) Biomasa de raíces por estaca. Los valores
corresponden a las medias ajustadas ± EE (n=50). Los datos fueron analizados
mediante Modelos Lineales Mixtos Generales o Generalizados. Letras diferentes
indican diferencias significativas entre tratamientos según el test post-hoc
DGC (α = 0,05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter 64S1,
IBA: ácido indol-3-butírico, LB: medio de cultivo Luria Broth, water: agua de red.
Figure
2. Rootstock 1103 Paulsen.
Figura 2. Portainjerto
1103 Paulsen.
101-14 MGt
The scion-rootstock union percentage between Malbec and 101-14
MGt rootstock decreased by 25% when cuttings were incubated with IBA compared
to the other treatments (figure 3A). Callus percentage
(~100%, p>0.05) (data not shown), rooting percentage (~100%), and root
biomass of 101-14 MGt were not affected by the treatments (figure
3B and 3D). The IBA treatment increased the number of roots per cutting (20
roots per cutting) compared to the water control (10 roots per cutting) (figure 3C), while 42P4 and LB treatments decreased this variable
to 7 roots per cutting compared to the water control. Inoculation of 101-14
rootstock cuttings with the 42P4 native strain did not promote rooting or graft
union.
A)
Scion-rootstock union percentage, B) Rooting percentage, C) Number of roots per
cutting, and D) Root biomass per cutting. Values correspond to adjusted means ±
SEM (n=50). Data were analyzed with General or Generalized Linear Models.
Different letters indicate significant differences between treatments according
to the post-hoc test DGC (α = 0.05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter
64S1, IBA: Indole-3-butyric acid, LB: Luria Broth culture medium, water:
tap water.
A) Porcentaje de
unión entre la púa y el portainjerto, B) Porcentaje de enraizamiento, C) Número
de raíces por estaca y D) Biomasa de raíces por estaca. Los valores
corresponden a las medias ajustadas ± EE (n=50). Los datos fueron analizados
mediante Modelos Lineales Mixtos Generales o Generalizados. Letras diferentes
indican diferencias significativas entre tratamientos según el test post-hoc
DGC (α = 0,05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter 64S1,
IBA: ácido indol-3-butírico, LB: medio de cultivo Luria Broth, water: agua de
red.
Figure
3. Rootstock 101-14 MGt.
Figura 3. Portainjerto
101-14 MGt.
110 Richter
Graft union percentage between Malbec and 110 R rootstock was
higher in autoclaved LB compared to the other treatments (figure
4A). However, callus percentage (~100%, p > 0.05, data not shown),
rooting percentage (~75%), and root biomass per cutting were similar among
treatments (figure 4B and 4D). Root number per cutting was
higher in cuttings incubated with IBA (4 roots per cutting) than in other
treatments (2.5 roots per cutting) (figure 4C). Pseudomonas
42P4 did not promote rooting or graft union of 110 R.
A)
Scion-rootstock union percentage, B) Rooting percentage, C) Number of roots per
cutting, and D) Root biomass per cutting. Values correspond to adjusted means ±
SEM, n=50. Data were analysed with General or Generalized Linear Models.
Different letters indicate significant differences between treatments according
to the post-hoc test DGC (α = 0.05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter
64S1, IBA: Indole-3-butyric acid, LB: Luria Broth culture medium, water:
tap water.
A) Porcentaje de
unión entre la púa y el portainjerto, B) Porcentaje de enraizamiento, C) Número
de raíces por estaca y D) Biomasa de raíces por estaca. Los valores
corresponden a las medias ajustadas ± EE (n=50). Los datos fueron analizados
mediante Modelos Lineales Mixtos Generales o Generalizados. Letras diferentes
indican diferencias significativas entre tratamientos según el test post-hoc
DGC (α = 0,05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter 64S1,
IBA: ácido indol-3-butírico, LB: medio de cultivo Luria Broth, water: agua de
red.
Figure
4. Rootstock 110 Richter.
Figura 4. Portainjerto
110 Richter.
SO4
Union percentage between Malbec and SO4 was higher in cuttings
incubated with 42P4 compared to the other treatments (50% higher than IBA and
water, and 25% higher than the LB treatment) (figure 5A).
Callus percentage (~80%, p>0.05) and rooting percentage (40%) of the SO4
rootstock were similar among treatments (figure 5B). Root
number and biomass per cutting were higher in cuttings incubated with IBA than
in other treatments (figure 5C and 5D). Pseudomonas 42P4
did not promote rooting but increased scion-rootstock union percentages.
A)
Scion-rootstock union percentage, B) Rooting percentage, C) Number of roots per
cutting, and D) Root biomass per cutting. Values correspond to adjusted means ±
SEM (n=50). Data were analysed with General or Generalized Linear Models.
Different letters indicate significant differences between treatments according
to the post-hoc test DGC (α = 0.05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter
64S1, IBA: Indole-3-butyric acid, LB: Luria Broth culture medium, water:
tap water.
A) Porcentaje de
unión entre la púa y el portainjerto, B) Porcentaje de enraizamiento, C) Número
de raíces por estaca y D) Biomasa de raíces por estaca. Los valores
corresponden a las medias ajustadas ± EE (n=50). Los datos fueron analizados
mediante Modelos Lineales Mixtos Generales o Generalizados. Letras diferentes
indican diferencias significativas entre tratamientos según el test post-hoc
DGC (α = 0,05). 42P4: Pseudomonas 42P4, 64S1: Enterobacter 64S1,
IBA: ácido indol-3-butírico, LB: medio de cultivo Luria Broth, water: agua de
red.
Figure
5. Rootstock SO4.
Figura 5. Portainjerto
SO4.
Discussion
This study is the
first to report applications of native PGPR strains from drylands in grapevine
propagation. We found that a Pseudomonas PGPR can improve rooting in
vine woody cuttings. We evaluated the ability of two native PGPR from arid
zones, Pseudomonas 42P4 and Enterobacter 64S1, to stimulate
rooting of ungrafted and grafted Vitis woody cuttings. Concerning
Malbec’s rooting, Pseudomonas 42P4 improved rooting compared to the
control, matching IBA results (figure 1, and figure
6).
Chart
sectors show the evaluated variables: rooting percentage, number of roots, and
root biomass per cutting. Green indicates significant promotion of the
parameter compared to the water control. Red indicates lower values, and yellow
indicates no significant differences among means. 42P4: Pseudomonas 42P4,
64S1: Enterobacter 64S1, IBA: indole-3-butiric acid, water: tap water.
Los
sectores del gráfico muestran las variables evaluadas: porcentaje de
enraizamiento, número de raíces y biomasa de raíces por estaca. El color verde
indica que el tratamiento promovió significativamente el parámetro en
comparación con el control con agua; el color rojo indica una disminución en
los valores observados, y el color amarillo indica que no hubo diferencias
significativas entre las medias. 42P4: Pseudomonas 42P4, 64S1: Enterobacter
64S1, IBA: ácido indol-3-butírico, wáter: agua de red.
Figure
6. Malbec. Coloured rings represent the treatments
(water, IBA, 42P4, and 64S1).
Figura
6. Malbec. El gráfico representa a los
tratamientos (agua, IBA, 42P4 y 64S1) como anillos.
Conversely, Enterobacter
64S1 failed to promote rooting of Malbec cuttings. Concerning grafted
material, Pseudomonas 42P4’s promoted rooting and graft union in a
rootstock-dependent fashion, enhancing rooting in 1103 Paulsen, but not
affecting the other rootstocks (figure 2, figure
3, figure 4, figure 5 and figure
7).
Optimal auxin concentrations stimulate adventitious rooting,
while higher concentrations are inhibitory (Garay-Arroyo
et al., 2014). The root-promoting effect of Pseudomonas 42P4
on Malbec own-rooted woody cuttings could be attributed to IAA production by
this bacterium (Perez-Rodriguez et al., 2020a).
Each
chart represents a treatment (water, IBA, 42P4) as rings. Chart sectors show
the evaluated variables rooting percentage, number of roots, and root biomass
per cutting. Green indicates significant promotion of the parameter compared to
the water treatment; red indicates lower values, and yellow indicates no
significant differences among means. 42P4: Pseudomonas 42P4, IBA:
indole-3-butiric acid, water: tap water.
Cada gráfico
representa los tratamientos (agua, IBA, 42P4) como anillos. Los sectores del
gráfico muestran las variables evaluadas: porcentaje de enraizamiento, número
de raíces y biomasa de raíces por estaca. El color verde indica que el
tratamiento promovió significativamente el parámetro en comparación con el
tratamiento con agua; el color rojo indica una disminución en los valores
observados, y el color amarillo indica que no hubo diferencias significativas
entre las medias. 42P4: Pseudomonas 42P4, IBA: ácido indol-3-butírico,
wáter: agua de red.
Figure
7. Rootstocks A) 1103 Paulsen, B) 101-14 MGt, C) 110
Richter and D) SO4.
Figura 7. Portainjertos
A) 1103 Paulsen, B) 101-14 MGt, C) 110 Richter y D) SO4.
However, as Enterobacter
produces an in vitro higher concentration of auxins than Pseudomonas
42P4 (six times more) (Perez-Rodriguez et al.,
2020a), an excessive concentration of IAA may have led to an inhibitory
effect. Considering this, we had previously tested lower Enterobacter dilutions
(104, 105
and 106 CFU mL-1)
and observed no rooting promotion. Another plausible explanation is the plant
recognizing Enterobacter as a pathogen. However, this would indicate a
species-specific response. In this regard, we previously reported Enterobacter
promoted growth in pepper and tomato (Perez-Rodriguez
et al., 2020a; Lobato Ureche et al., 2021).
Rooting capacity
varied with rootstock (Keller 2020; Ollat et al.,
2016). We found that 101-14 MGt showed the highest rooting compared with
the control treatment, followed by 1103 P and 110R, while SO4 had the lowest
rooting (figure 2, figure 3, figure
4, and figure 5). SO4 (Berlandieri-Riparia family),
1103 P (Berlandieri-Rupestris family), and 110R (Berlandieri-Rupestris
family) were expected to have lower rooting capacity than 101-14 MGt (Riparia-Rupestris
family), because they are hybrids of V. berlandieri, an American
difficult-to-root Vitis species (Keller et al.,
2020; Riaz et al., 2019). Cuttings of 1103 P incubated with Pseudomonas
42P4 showed increased rooting compared to the control. Additionally,
rooting percentage was 15% higher than with the synthetic rooting agent IBA,
which only promoted two of the four assessed parameters. However, contrasting
reports exist regarding IBA’s effectiveness in 1103 P (Boeno
et al., 2023; Daskalakis et al., 2018; Gordillo et al.,
2022; Satisha et al., 2008). Rooting of 110 R, 101-14, and SO4
cuttings inoculated with Pseudomonas showed no significant improvements
compared to the control. Isçi et al. (2019)
evaluated a commercial bacteria consortium on Ramsey rootstock after nursery
forcing, successfully increasing rooting percentage and root DW compared to
IBA. Rootstock response to Pseudomonas 42P4 and IBA treatments may
depend on genetic diversity and the presence of inhibitors (Wilson and Van Staden, 1990).
Scion-rootstock
union percentage in SO4 cuttings inoculated with Pseudomonas 42P4 was
threefold higher than in water or with IBA (75% vs. 25%). This result aligns
with Köse et al. (2005), who reported that Pseudomonas
BA8 promoted graft union of the Italia and Beyaz Çavuş scions onto 41B and
5BB rootstocks. Similarly, Toffanin et al. (2016)
evaluated the effect of inoculating nine rootstock cuttings with Azospirillum
brasilense Sp245 and found improved union only in 1103 P grafted onto
Sangiovese. Likewise, A. brasilense Sp245 only improved the union of
Colorino grafted onto 420A (Bartolini et al.,
2017). Our native Pseudomonas’ failure to improve graft union in
three out of the four evaluated rootstocks could be explained by impaired polar
auxin transport. Auxins may not have moved towards the rootstock-scion union,
but rather accumulate at the cutting’s basal end, probably given its genetic
origin. V. riparia and V. berlandieri develop callus on both
extremities, while V. rupestris predominantly forms it on the upper
extremity (Galet, 1993).
Considering the Vitis genus cuttings lack preformed root
primordia, adventitious root formation is a prerequisite for successful cutting
propagation (Hartmann et al., 2014). Once
cuttings are removed from the plant (wounding), a series of wound responses
occur, and de novo adventitious root generation proceeds. De novo adventitious
rooting involves four stages: cell dedifferentiation (possibly medullary rays
in grapevine), cell proliferation, development and organization of root
primordia, and growth of root primordia (Hartmann et
al., 2014). In early rooting stages, high auxin concentrations either
from buds or exogenous sources like IAA are necessary for dedifferentiation and
cell proliferation (Hartmann et al., 2014;
Jarvis, 1986). Under our conditions, the native Pseudomonas synthetised
and exuded IAA into the medium where bacteria had grown and cuttings were
incubated. Although we do not know whether the strains can colonise cuttings
endophytically or epiphytically, we believe bacteria may have produced IAA in
the medium, promoting rooting. Currently, IBA and NAA are the most commonly
used auxins in nurseries (Waite et al., 2014).
Synthetic growth regulators often excessively used, can be sufficiently
replaced by organic products for plant rooting, at least for some species and
varieties (Atak et al., 2024). However,
further investigation should consider the mechanisms underlying bacterium-plant
interaction between Pseudomonas 42P4 and Vitis spp. and long-term effects.
Conclusions
Pseudomonas 42P4, but not Enterobacter 64S1
promoted rooting (rooting percentage, root number and biomass per cutting) of
Malbec cuttings reaching similar values as IBA 1000 ppm. Furthermore, this
strain increased 1103 P rooting (rooting percentage, root number and biomass
per cutting), compared to the water treatment, but. This was not observed in
101-14 MGt, 110 R or SO4. In 1103 P, this strain increased rooting percentage,
exceeding IBA treatment. Pseudomonas 42P4 also increased SO4 graft union
threefold compared to IBA and water. The use of this bacterium, native to arid
soils, enhances rooting parameters of ungrafted and grafted V. vinifera cv.
Malbec cuttings. This capacity of Pseudomonas 42P4 presents a promising
sustainable alternative for improving grapevine production in commercial nurseries.
Acknowledgements
The authors would
like to thank Diana Segura, Micaela Perez-Rodriguez, Martín López, Walter Tulle
and Carlos Blanquer for their assistance with morphological measurements.
This study was
supported through funding from Fondo para la Investigación Científica y
Tecnológica de Argentina (FONCYT, PICT 2020-3618 to ACC); Universidad Nacional
de Cuyo (SIIP-UNCUYO M021-T1 to CVG and ACC), Fundación Williams (to ACC) and
CONICET (PIP-KA1 11220210100195CO to ACC and CVG).
ACC and CVG are CONICET researchers and UNCUYO Professors, MGG
is a PhD fellow from CONICET and FC is a member of Trapiche winery staff.
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