Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Tomo 56(2). ISSN (en línea) 1853-8665. Año 2024.

Original article

First report of the causal agent of vine crown gall in Mendoza, Argentina

Primer reporte del agente causal de la agalla de corona de la vid en Mendoza, Argentina

Sandra D’Innocenzo^1*,

Mariano Emanuel Diaz¹,

Maria Georgina Escoriaza^{2, 3}

¹INTA EEA Mendoza. Laboratorio de Fitopatología. San Martín 3853. Luján de Cuyo. C. P. 5507.

²Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Cátedra de Industrias Agrarias FCA UNCuyo. Almirante Brown 500. M5528AHB. Chacras de Coria. Mendoza. Argentina.

³INTA EEA La Consulta. Laboratorio de Fitopatología.

* dinnocenzo.sandra@inta.gob.ar

Abstract

Crown gall is one widespread grapevine disease in the world, caused by Allorhizobium vitis (syn. Agrobacterium vitis) and Agrobacterium tumefaciens (syn. Rhizobium radiobacter). All. Vitis, is the predominant species and primary cause of the disease. This study aimed to identify and characterize molecularly the agrobacteria present in plants with crown gall symptoms in Mendoza vineyards. Diseased plants with trunk trunk-based galls were sampled from different areas of Mendoza. For bacterial identification and characterization, two multiplex PCRs were performed demonstrating that 91.6% of the strains obtained were agrobacteria (77% A. tumefaciens and 23% All. vitis). Fifty percent of All. vitis and 16% of A. tumefaciens identified strains were pathogenic. Pathogenicity tests were also conducted on Kalanchoe daigremontiana, with resulting tumorigenic symptoms.

Keywords: Allorhizobium, Agrobacterium, Vitis vinifera, crown gall, Mendoza

Resumen

Una de las enfermedades de la vid ampliamente distribuida en el mundo es la agalla de corona, que tiene como agente causal a Allorhizobium vitis (syn. Agrobacterium vitis) y Agrobacterium tumefaciens (syn. Rhizobium radiobacter), siendo la primera especie la que predomina como agente causal de la enfermedad en vid. El objetivo de este estudio fue identificar mediante técnicas moleculares las agrobacterias patógenas presentes en plantas con síntomas y determinar cuál de ellas predomina en viñedos de la provincia de Mendoza. Plantas de vid con agallas en el tronco provenientes de diversas zonas de la provincia de Mendoza se utilizaron para realizar los aislamientos. Para la identificación y caracterización molecular de los aislados se realizaron dos reacciones múltiples de PCR. Se identificó el 91,6% de las cepas obtenidas como agrobacterias (77% A. tumefaciens y 23% All. vitis). Se determinó que el 50% del total identificado como All. vitis son cepas patógenas, mientras que para A. tumefaciens sólo el 16% de los aislados dio patogenicidad positiva. También se realizaron pruebas de patogenicidad en Kalanchoe daigremontiana, donde se observó el desarrollo de los síntomas típicos de tumorigénesis.

Palabras clave: Allorhizobium, Agrobacterium, Vitis vinifera, agalla de corona, Mendoza

Originales: Recepción: 03/05/2024 - Aceptación: 04/09/2024

Introduction

With 207,047 hectares cultivated with grapevines (Vitis vinífera), Argentina leads the international wine industry. The province of Mendoza produces 70% of Argentinian wine ⁽¹¹⁾ and is considered one of the Wine Capitals Worldwide. This industry, including grape growing, wine and must production, and tourism, is fundamental to the economic development of the province.

Various pests and diseases significantly reducing production quantity and quality affect grapevine cultivation. Crown gall, a disease caused by Allorhizobium vitis ⁽¹⁵⁾ and Agrobacterium tumefaciens ⁽¹⁴⁾, is among the most important and widespread vine diseases globally. These bacteria were first isolated in the United States in 1907 and were later reported in China, Japan, South Africa, and some countries in Europe and South America ⁽⁴⁾.

In grapevine, All. vitis is the predominant species causing the disease, while A. tumefaciens is found less frequently and in smaller proportions. A. tumefaciens is polyphagous and can affect several dicotyledon species, including Solanaceae and various Asteraceae ^{(4, 6)}. Currently, rrs analysis and constitutive genes have described new species of Agrobacterium initially identified as A. tumefaciens in various hosts ⁽⁷⁾.

A. tumefaciens and All. vitis exist in nature as pathogenic and non-pathogenic strains. Pathogenic strains contain a non-essential tumor-inducing plasmid (pTi) involved in disease triggering ⁽¹⁶⁾. All. vitis genomic organization is characterized by two circular chromosomes. The smaller, chromosome II later classified as a chromid, is essential for disease development. A. tumefaciens carries one circular chromosome and a secondary linear chromid ⁽¹⁶⁾.

Typically, the process begins with a wound in the trunk or roots. The wound releases chemical signals that, perceived by bacteria, induce virulence ⁽²⁾. The disease is triggered when certain genes from the Ti plasmid are transferred to the host genome, encoding overexpression of phytohormone synthesis. This overexpression augments cell division (hyperplasia) and cell size (hypertrophy), leading to the characteristic tumor. This plasmid also contains genes encoding opine synthesis. Opines are low-molecular-weight compounds, used by agrobacteria as carbon and nitrogen source ⁽¹⁸⁾. According to Kuzmanović et al. (2020). Ti plasmids are classified into three major groups: octopine, nopaline and vitopine. Genes coding for octopine are present in 60% of the strains. About 30% of strains carry the nos genes (nopaline synthase), and only 10% of strains have vitopine type ⁽⁴⁾.

The development of one or more tumors around a diseased organ alters sap movement, causing chlorosis, vigor loss and decreased production. In extreme cases, it may lead to plant death, including nursery young plants or cuttings ⁽⁴⁾.

Several chromosomal genes aid in accurate identifications of pathogenic agrobacteria species. Plasmid genes determine the presence of pathogenicity-related oncogenes. Due to the importance of viticulture in Mendoza, this study aimed to define the main molecular traits of the causal agent of crown gall identifying pathogenic species. We finally aimed to determine the predominant species in Mendoza vineyards.

Materials and methods

Plant samples and Bacterial strains

One hundred and forty-eight symptomatic plants (figure 1) were collected from various vine-growing areas of Mendoza (figure 2).

Figure 1. Symptoms of crown gall on Mendoza grapevines.

Figura 1. Síntomas de agalla de corona en vides de Mendoza.

Figure 2. Mendoza, sampled departments.

Figura 2. Mendoza, departamentos muestreados.

Composite samples were taken from plants within the same vineyard, resulting in 86 samples to be analyzed (table 1).

Table 1. Strain identification, geographical origin, plant age, cultivar, number of plants, number of analyzed samples, isolation.

Tabla 1. Identificación de la cepa, origen geográfico, edad de las plantas, cultivar, número de plantas, número de muestras analizadas y aislamiento.

UN: unknown/desconocido.

Galls were washed with running water. Subsequently, they were disinfected with 1.1% sodium hypochlorite for 5 minutes in a laminar flow cabinet. After disinfection, they were rinsed twice with sterile distilled water, completely removing sodium hypochlorite. Galls were then cut into small pieces, discarding the external part to minimize contaminating microorganisms. The resulting pieces were placed in 5 ml sterile distilled water for one hour allowing diffusion of bacteria in the sample.

Each bacterial suspension was streaked onto Roy and Sasser (RS) semiselective culture medium for All. vitis and Schroth culture medium for A. tumefaciens. Culture plates were incubated at 27 °C in darkness. Colony development was observed after seven days. All. vitis colonies on RS medium had a dark red center with transparent or white edges. The red center is not always evident (Schaad et al., 2001 and Burr, T. J. personal communication, June 28, 2016). A. tumefaciens colonies acquire a reddish color in Schroth culture medium. Colonies with these characteristics were then transferred to Luria Bertrani (LB) culture medium.

DNA extraction and specific PCR amplification

DNA was extracted according to Khlaif, H. and Al-Karablieh (2002). All. vitis and A. tumefaciens, were differentiated according to differences in the 23S rDNA gene ⁽²⁰⁾. A universal forward and two specific reverse primers were used: B1R for A. tumefaciens and AvR for All. vitis (table 2).

Table 2. Primers for identification and molecular characterization of All. vitis and A. tumefaciens strains.

Tabla 2. Primers usados para identificación y caracterización molecular de cepas de All. vitis y A. tumefaciens.

Multiplex PCR (polymerase chain reaction) used the following reagents: 1X PCR buffer, 1.5 mM MgCl2, 200 mM dNTP, 1 mM of each primer and 1U of Recombinant DNA polymerase (Invitrogen) and 5 μl of template DNA for a final reaction volume of 25 μl. The PCR consisted of initial denaturation at 94°C 1 min, 35 cycles at 94°C 1 min, 67°C 1 min, 72°C 1.5 min and 72°C 10 min, using an Eppendorf thermocycler.

Multiplex PCR with specific primers for oncogenes allowed for pathogenic strain detection. The reaction combined the primers iaaHF2/iaaHR1 and S4iaaM5/S4iaaM3 for the auxin-biosynthesis genes iaaH and iaaM, respectively. The reaction was carried out in a final volume of 25 μl, with 1X Buffer, 1.5 mM MgCl2, 0.5 μM of each primer, 200 μM of dNTP, 1.25 U of polymerase (Invitrogen Platinum DNA polymerase) and 1 μl of DNA. Amplification began with initial denaturation at 94°C for 1 min, followed by 30 cycles at 92°C 1 min, 54°C 1 min, 72°C 1.5 min and 72°C 3 min ⁽³⁾.

PCR-generated amplicons were detected by electrophoresis using 1% agarose gel, run at 90 volts for 1 hour and stained with ethidium bromide. The gels were visualized under UV light and photo-documented using Bio-Rad equipment and Quantity One software. Band size was compared with a 100 bp ladder molecular marker (Invitrogen).

Pathogenicity tests

PCR results were confirmed via biological tests performed on Kalanchoe daigremontiana plants to evaluate isolate-pathogenicity. Inoculation was carried out through punctures on the stem with a micropipette tip and 2.5 μl of bacterial suspension 109 cfu/ml of each strain. Each strain was inoculated in three plants via 5 stem wounds per plant. Sterile distilled water was the negative control and All. vitis and A. tumefaciens reference strains were positive controls.

The plants were kept in the laboratory at room temperature, and covered with plastic bags to maintain approximately 90 % humidity for three days. Then, bags were removed and plants were taken to the greenhouse. Observations were made every 15 days for two months ⁽²³⁾. Isolations from tissues developed in the inoculation zone were carried out in a semiselective culture medium, using the same method as with vine galls.

Results

Molecular analysis

Sixty-nine isolates out of 86 samples analyzed resulted in 91.6% identified as agrobacteria, among which 77% were A. tumefaciens and 23%. All. vitis. Figure 3 presents a PCR with 3 isolates where A1 was A. tumefaciens and A2 and A3 were All. vitis.

From left to right: M: marker 100 bp (PROMEGA); Bco: water; At: A. tumefaciens reference strain; Av: All. vitis reference strain.; gall isolates: A1, A2, A3.

De izquierda a derecha: M: marcador 100 pb (PROMEGA); Blanco: agua; At: cepa de referencia de A. tumefaciens; Av: cepa de referencia de All. vitis; aislados de agalla: A1, A2, A3.

Figure 3. Multiplex PCR with primer pairs UF/B1R (184 bp) and UF/AvR (478 bp).

Figura 3. PCR múltiple con los pares de primers UF/B1R (184 pb) y UF/AvR (478 pb).

The multiplex PCR was performed with the combination of primers iaaHF2/iaaHR1, while S4iaaM5/S4iaaM3 determined pathogenicity. The iaaH gene was only amplified on 16% of A. tumefaciens strains, molecularly identified as pathogenic, and 50% of All. vitis isolates proved to be pathogenic. The iaaM gene did not amplify. Figure 4 shows amplification of the pathogenicity gene present in both species. Isolates A1 of A. tumefaciens and A2 and A3 of All. vitis present positive pathogenicity.

From left to right: M: 100bp marker (Promega), Bco: water; At: A. tumefaciens reference strain; Av: All. vitis reference strain; gall isolates: A1, A2 and A3.

De izquierda a derecha: M: marcador 100 pb (Promega), Blanco: agua; At: cepa de referencia de A. tumefaciens; Av: cepa de referencia de All. vitis; aislados de agalla: A1, A2 y A3.

Figure 4. Multiplex PCR with primer pairs iaaHF/iaaHR (420 bp) and S4iaaM5/S4iaaM3 (800 bp).

Figura 4. PCR múltiple con los pares de primers iaaHF/iaaHR (420 pb) y S4iaaM5/S4iaaM3 (800 pb).

Pathogenicity Test

Two weeks after inoculation, positive results were observed in Kalanchoe plants. Abnormal growth and color change (redness) were similar to those in plants inoculated with reference strains. In some cases, corky tissue developed at the inoculation site (figure 5). These results became more pronounced two months after inoculation.

A: Negative control (water), B: reference strain of A. tumefaciens, C: reference strain of All. vitis, D, E and F: gall isolates: A1, A2 and A3 with positive pathogenicity result (molecular analysis).

A: Control negativo (agua), B: cepa de referencia de A. tumefaciens, C: cepa de referencia de All. vitis, D, E y F: aislado muestra con resultado de patogenicidad (análisis molecular) positivo.

Figure 5. Symptoms observed in Kalanchoe stems 2 weeks after inoculation.

Figura 5. Síntomas observados en tallos de Kalanchoe a las 2 semanas de la inoculación.

Discussion

Bacterial genetic diversity of both species limits detection efficiency in grapevines ⁽³⁾. All. vitis strains are genetically diverse (4; 8; 9; 16; 23). Our data suggest All. vitis could be a species complex comprising several genomic species ⁽¹⁶⁾. In this study, after obtaining pure and simple isolates, successful species identification followed the molecular protocol described by Pulawska et al. (2006). However, since this PCR does not identify pathogenicity genes, the analysis must be complemented with additional PCR determining gene presence ^{(1, 3, 8, 17, 19, 22)}. This research used specific primers iaaHF2/iaaHR1 and S4iaaM5/S4iaaM3 for iaaH and iaaM genes, respectively, showing non-pathogenic A. tumefaciens strains predominated over All. vitis strains in the analyzed grapevine samples. However, 50% of All. vitis isolates were pathogenic. This finding indicates that All. vitis is the predominant pathogenic species and main disease cause in grapevines studied in Mendoza, in agreement with prior studies ^{(2, 5, 10, 14, 22)}.

The same PCR determining pathogenicity, determined opine type. We found absent vitopina type in all All. vitis isolates and presence of the octopine/nopaline types in Mendoza,

Seventy-nine percent of pathogenicity tests in inoculated Kalanchoe showed disease symptoms. This value is within the expected range (78-94%) ⁽²¹⁾. These data also align with Kuzmanović et al. (2016), who observed that some strains did not demonstrate their tumorigenic capacity in inoculated plants despite possessing pathogenicity-associated genes molecularly identified. This suggests that such isolates remain potentially tumorigenic. However, pathogenicity is influenced by plant age and environmental conditions. Absent Crown gall symptoms do not imply absent tumorigenesis genes ⁽¹⁸⁾, probably because no single host is infected by more than 81% of pathogenic strains and not all strains produce tumors in every host ⁽¹³⁾. According to Lamovšek et al. (2014), determining pathogenicity through molecular tests might replace biological tests. The PCR are less time-consuming and labor-intensive. However, given the occurrence of false negatives, pathogenicity tests remain a valuable tool in plant bacteriology.

Conclusions

This study successfully identified and characterized the causal agents of Crown gall in Mendoza vineyards using molecular methods. Our methodology enables the characterization of agrobacteria in Argentina and provides a quick and precise diagnostic tool, even for evaluating asexually propagated grapevines.

This information will help develop management strategies to reduce disease spread and incidence in our vineyards and nurseries and improve the health and productivity of their vineyards.

Finally, our results aiding bacterial identification in plant material allow for protocols to detect bacteria in asymptomatic material ensuring propagation of healthy plants from health-controlled material.

To the best of our knowledge, this is the first study identifying uncited crown gall species in Argentina.

Acknowledgment

Dr. Ibrahim Tolba (Plant Pathologist at the Faculty of Agriculture, Ain Shams University, Egypt) for supplying positive controls.

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