An efficient inoculation method of Ralstonia ...

Trop. plant pathol. DOI 10.1007/s40858-015-0056-2

SHORT COMMUNICATION

An efficient inoculation method of Ralstonia solanacearum to test wilt resistance in Eucalyptus spp. Natália R. Fonseca 1 & Leonardo S. S. Oliveira 1 & Lúcio M. S. Guimarães 1 & Ramon U. Teixeira 1 & Carlos A. Lopes 2 & Acelino C. Alfenas 1

Received: 21 July 2015 / Accepted: 6 October 2015 # Sociedade Brasileira de Fitopatologia 2015

Abstract The variation in disease incidence of bacterial wilt caused by Ralstonia solanacearum among eucalypt clones (Eucalyptus spp.) in the field indicates that the disease may be controlled by planting resistant material. However, efficient inoculation methods for bacterial wilt on eucalypt are scarce and have low replicability. In this work, we developed an effective protocol for inoculation of R. solanacearum, which was subsequently validated on different eucalypt clones. Three methods were tested: (i) soil infestation with bacterial cell suspension; (ii) immersion of wounded roots in the bacterial cell suspension; and (iii) injection of bacterial cell suspension in the base of the stem. The injection method proved to be the most efficient for inoculating eucalypt with R. solanacearum. Differentiation between resistant and susceptible clones was observed 30 days after inoculation in independent assays. Base stem inoculation of 21 eucalypt clones showed that four clones, classified as resistant, did not exhibit wilt symptoms or bacterial ooze at the end of the experiment. Although no wilting symptoms were observed, four other clones were considered susceptible because at least one plant showed bacterial ooze from the inoculated tissue. The remaining 13 clones were highly susceptible, presenting typical wilt symptoms and bacterial ooze. Keywords Wilt disease . Bacterial ooze . Eucalyptus breeding Section Editor: F. Murilo Zerbini * Acelino C. Alfenas [email protected] 1

Dep. de Fitopatologia, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil

2

Embrapa Hortaliças, Caixa Postal 218, 70359-970 Brasília, DF, Brazil

Ralstonia solanacearum (Smith) Yabuuchi et al. (1995) is a destructive bacterial plant pathogen that causes bacterial wilt disease on many plant species. In Brazil, bacterial wilt was first reported on eucalypts in the early 1980’s in the region of Prata, Minas Gerais state (Sudo et al. 1983). Subsequently, the disease was observed in the states of Bahia, Pará, Espírito Santo, Maranhão, Santa Catarina and Goiás (Fonseca et al. 2014). In addition to Brazil, the disease has been reported in China (Wu and Liang 1988a), Taiwan (Wang 1992), Venezuela (Ciesla et al. 1996), Thailand (Pongpanich 2000), Vietnam (Thu et al. 2000) and Paraguay (Santiago et al. 2014). In Africa, the disease has been detected in eucalypt plantations in South Africa, the Democratic Republic of Congo and Uganda (Roux et al. 2001). In mini-clonal hedges, the disease is characterized by leaf necrosis, which includes darkening of the stem base, complete discoloration of internal tissue, wilting and eventual root death. Symptoms in the shoots are similar to the gradual death of mini-stumps that are subjected to drastic pruning or have a malformed root system (Alfenas et al. 2006). In the rooting stage, infected cuttings may exhibit reddening of leaves and subsequent rot. According to Mafia et al. (2012), the spread of the pathogen and disease transmission may occur during the cloning process. In 2005, bacterial wilt caused significant losses in several eucalypt clonal nurseries in Brazil, totaling an estimated loss of at least US$ 27 million (Alfenas et al. 2009). In the field, the disease is characterized by reddening, wilt, leaf spot, ascending basal leaf loss, internal discoloration of the wood and eventual plant death, which usually occurs in the fourth month following planting (Alfenas et al. 2006). After detection of the pathogen in planting areas, disease management is difficult and genetic resistance is the most viable option. However, previous studies focusing on the development of artificial inoculation methods rarely showed reliable reproduction of the disease’s symptoms (Cruz and

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Dianese 1986; Dianese et al. 1990; Dianese and Dristig 1993; Coutinho et al. 2000), or required a specific facility using infected sand (Mafia et al. 2014). In a recent study using four different inoculation methods, Wei et al. (2014) were able to reproduce the symptoms of the disease by injection of inoculum into the root collar. However, they pointed out the need of improving this method or developing a more efficient inoculation procedure. Thus, the objective of this work was to develop a simple and efficient inoculation method of R. solanacearum for eucalypt, and subsequently validate the inoculation method using different eucalypt clones. In all experiments we used R. solanacearum strain UFV32, which caused high levels of disease intensity on eucalypt in previous experiments. This strain was isolated from E. grandis in the region of Eunápolis, Bahia state (Alfenas et al. 2006) and characterized as belonging to biovar 1 and phylotype IIA (Fonseca et al. 2014). To evaluate the effectiveness of each inoculation method, 30 plants of Clone V (E. urophylla) were used, which in preliminary tests showed susceptibility to bacterial wilt (data not shown). Ninety-day-old cuttings were used for the three methods. With the exception of the method using wounded roots immersed in bacterial suspension, the cuttings were transplanted to 2 L pots that contained the commercial substrate MecPlant supplemented with 6 kg/m3 of superphosphate and 3 kg/m3 Osmocote (6-19-10). Cuttings were inoculated 20 days after transplanting. One tomato plant (Solanum lycopersicum L. cv. Moneymaker) was used as an indicator of the strain’s virulence for each method. Before inoculation, colonies of the strain UFV32 were grown in Petri dishes (90×15 mm) containing Kelman media (Kelman 1954) and then incubated at 28 °C. After 48 h, smooth, fluid colonies of white edges and red centers were transferred to new plates containing 523 medium (Kado and Heskett 1970) and incubated for 24 h at 28 °C. Following incubation, 0.85 % sodium chloride (w/v) was added to the plates and the bacterial colonies were removed by scraping the surface using a Drigalski spatula. The concentration of the bacterial inoculum suspension was adjusted spectrophotometrically (A540 =1.0, approximately 1×109 CFU/mL). Three procedures were evaluated to identify the best inoculation method for R. solanacearum on eucalypt: (1) soil infestation with bacterial cell suspension; (2) immersion of wounded roots in bacterial cell suspension; and (3) injection of bacterial cell suspension in the base of the stem (Fig. 1). Method 1—Soil infestation with R. solanacearum strain UFV32: The soil was infested by adding 100 mL of the bacterial cell suspension around the stem base of each plant. Method 2—Immersion of wounded roots in bacterial cell suspension: approximately one-third of the root system was removed from the eucalypt cuttings before transplantation. The roots were immersed in the bacterial suspension for 10 min. Subsequently, the plants were transplanted to 2 L pots.

Fig. 1 Inoculation methods of Ralstonia solanacearum in eucalypts. a Soil infestation with R. solanacearum cell suspension; b Immersion of wounded roots in bacterial cell suspension; c Injection of bacterial cell suspension at the stem base

Method 3—Injection of bacterial cell suspension at the base of the stem: plants were wounded by making a 3-mm deep and 10-mm long downward-slanting cut with a sterile scalpel in the outer bark into the wood at 5 cm above the ground line. A piece of damp cotton was placed below the stem cut to prevent loss of the inoculum and also to keep the inoculation site favorable to infection. The bacterial suspension (0.5 mL) was deposited into the wound. Following inoculation, the wound and the cotton were covered with Parafilm to prevent contamination by other pathogens and to maintain moisture in the chamber. For the three methods, the plants were kept in a growth chamber at 28±2 °C with a 12 h photoperiod and light intensity of 40 μmol/s/m2. For each method, five replicates of clonal cuttings were used per treatment in a completely randomized design. Five

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plants inoculated with 0.85 % sodium chloride instead of the bacterial suspension served as control treatments for each method. The experiment was carried out twice in different times of the year. Plants were evaluated daily for wilt symptoms and, 30 days after inoculation, for disease incidence and bacterial ooze. To evaluate bacterial ooze, five fragments of stem tissue of wilted or symptomless plants were removed from the plant 2 cm above the inoculation point, deposited on a microscope slide containing a drop of water and examined under a light microscope at 100× magnification. The injection of bacterial cells in eucalypt stems (method 3) was the only method capable of inducing typical symptoms of bacterial wilt on eucalypt cuttings of the Clone V (Table 1). When the disease developed, brown and dark discoloration was observed on the wounds of shoots. Plants exhibited wilting symptoms within 10 to 23 days after inoculation. Wilt symptoms initiated at the apex of the plant and moved towards the base, and defoliation was observed following the occurrence of wilting. Besides wilt symptoms, all five inoculated plants of Clone V displayed abundant bacterial ooze. Despite soil infestation representing a more natural inoculation method, no wilt symptoms or bacterial ooze were observed during the period of 30 days. This result is commonly observed for other susceptible hosts, such as tomato, and it is usually attributed to disease escape (Lopes and Boiteux 2012).

Table 1 Inoculation of Eucalyptus with Ralstonia solanacearum using three different methods

The removal of a portion of the root, used in method 2, although being a drastic inoculation method, was not sufficient to cause disease symptoms during the evaluation period. Control eucalypt plants remained asymptomatic throughout the experiment and did not exhibit any bacterial ooze for the three methods tested. Tomato plants used as susceptible controls showed typical wilt symptoms and dark discoloration of internal portions of the stem tissue for the three methods (Fig. 2). Bacterial wilt resistance was assessed on 21 commercial clones of Eucalyptus spp. (Table 2). The clones were inoculated by injecting the bacterial cell suspension at the base of the stem (method 3). Five clonal replicates per clone were inoculated with the strain UFV32. This number of replicates per each clone has been used routinely and successfully in our laboratory to access resistance of Eucalyptus spp. to Ceratocystis wilt caused by Ceratocystis fimbriata, leaf blight caused by Xanthomonas axonopodis and Calonectria leaf blight caused by Calonectria pteridis (Fonseca et al. 2010). The experiment was conducted as a completely randomized design with five replicate plants per treatment. Five plants inoculated with 0.85 % sodium chloride instead of bacterial suspension were used as negative controls. Clones that showed no symptoms of disease or bacterial ooze at 30 days after inoculation were considered resistant. The clones were

Inoculation method

Replicate

Wilt symptoms

Bacterial ooze

Method 1 (Soil infestation with R. solanacearum cell suspension)

1 2 3 4

– – – –

– – – –

5 Ca Tb 1

– – + –

– – + –

2 3 4 5 C T 1 2 3 4 5 C T

– – – – – + + + + + + – +

– – – – – + + + + + + – +

Method 2 (Immersion of wounded roots in bacterial cell suspension)

Method 3 (Injection of bacterial cell suspension at the base of the stem)

a

C=non-inoculated control plant of eucalypt Clone V

b

T=susceptible control of tomato seedling cv. Moneymaker

Trop. plant pathol. Fig. 2 Evaluation of resistance of eucalypt clones to Ralstonia solanacearum inoculated by injection of bacterial cell suspension at the base of the stem. a, b Eucalypt cuttings inoculated in a growth chamber at 28±2 °C. Red arrow indicates wilt symptoms; c Comparison between non-inoculated control and inoculated eucalypt cuttings; d Comparison between control and inoculated tomato plants; e Bacterial ooze in water from sections of infected eucalypt stem; f Bacterial ooze from an inoculated susceptible clone in slide containing water under a light microscope at 100× magnification

considered susceptible when at least one of the replicate plants exhibited bacterial ooze from the inoculated stem tissue. Clones that showed bacterial ooze accompanied by wilt symptoms were classified as highly susceptible. Of the 21 clones, four (clones A, B, C, and D) were considered resistant, as they displayed no wilt symptoms or bacterial ooze. Four other clones (clones E, F, G, and H) showed no wilt symptoms. However, bacterial ooze was observed in at least one of the five inoculated plants. These clones were considered susceptible, especially because they may serve as inoculum carriers as latent infections. The remaining clones were highly susceptible because of the presence of typical bacterial wilt symptoms and bacterial oozing (Table 2). All clones of Eucalyptus dunnii exhibited typical wilt symptoms and bacterial ooze. Even the hybrid clone that has E. dunnii as one of the parents was highly susceptible, indicating that this species may represent a problem for eucalypt cultivation in areas where the disease is present. For E. saligna, variation in resistance within the species was observed, showing a range of resistant and susceptible clones. Clone D of E. saligna behaved as resistant throughout the experiments.

The present study showed that injection of bacterial cells at the base of the stem was the most efficient method for infection of R. solanacearum in eucalypt cuttings. This inoculation method differs from those used by Wei et al. (2014), and provides reliable results confirmed with a set of 21 commercial clones. Bacterial ooze proved to be an effective way to determine the susceptibility of eucalypt clones to bacterial wilt disease. Wilt symptoms are simpler and faster to observe, but this needs to be accompanied by evaluation of bacterial ooze, otherwise the symptoms observed might be attributed to other biotic or abiotic factors. Therefore, four clones showing no bacterial ooze were considered as resistant. Eucalyptus spp. may present different levels of resistance to infection by R. solanacearum (Dianese et al. 1990). In China, the most susceptible species were E. tereticornis, E. urophylla and E. camaldulensis (Wu and Liang 1988). Additionally, Ciesla et al. (1996) reported that E. grandis, E. pellita, E. saligna and the hybrid E. grandis x E. urophylla are also susceptible. In South Africa, the disease has been reported only in the hybrid E. grandis x E. camaldulensis (Coutinho et al. 2000). The study by Wu and Liang (1988) in China indicated that E. grandis,

Trop. plant pathol. Table 2 Clone

Reaction of eucalypt clones to bacterial wilt caused by Ralstonia solanacearum inoculated by injection at the stem base Species

No. of plants with symptoms/ No. of inoculated plants

Phenotype

Wilt symptoms

Bacterial ooze

0/5 0/5 0/5

0/5 0/5 0/5

Resistant Resistant Resistant

Clone A Clone B Clone C Clone D Clone E Clone F Clone G Clone H Clone I Clone J Clone K Clone L Clone M Clone N

Eucalyptus spp.1 Eucalyptus spp.1 Eucalyptus spp.1 E. saligna Eucalyptus spp.1 Eucalyptus spp.1 Eucalyptus spp.1 Eucalyptus spp.1 E. saligna Eucalyptus spp.1 Eucalyptus spp.1 Eucalyptus spp.1 Eucalyptus spp.1 E. urophylla x E. grandis

1/5 0/5 0/5 0/5 0/5

0/5 1/5 1/5 1/5 4/5

Resistant Susceptible Susceptible Susceptible Susceptible

5/5 4/5 3/5 3/5 3/5 3/5

3/5 4/5 5/5 5/5 5/5 5/5

Highly susceptible Highly susceptible Highly susceptible Highly susceptible Highly susceptible Highly susceptible

Clone O Clone P

E. dunnii E. urophylla x E. maidenii

5/5 5/5

5/5 5/5

Highly susceptible Highly susceptible

Clone Q Clone R Clone S Clone T Clone U

E. urophylla x E. globulus E. urophylla x E. maidenii E. urophylla x E. dunnii E. dunnii E. dunnii

5/5 5/5 5/5 5/5 5/5

5/5 5/5 5/5 5/5 5/5

Highly susceptible Highly susceptible Highly susceptible Highly susceptible Highly susceptible

1

Open pollinated hybrid clones

E. urophylla, E. saligna, Corymbia citriodora and E. exserta in some provinces were resistant to infection by R. solanacearum. However, due to the high variability found in strains of this pathogen, the information generated with inoculation studies seems to be restricted to the country of origin, as the strains applied in each experiment are variable. In our study, from the results obtained with the validation of inoculation methods, we found that clone D of E. saligna behaved as resistant, showing that variability within the host species is an alternative to breeding programs focusing on disease resistance. However, a more detailed study involving a larger number of species, provenances, clones, as well as different bacterial strains, is necessary to better understand which eucalypt genotypes should be integrated into a breeding program focused on disease resistance to bacterial wilt. Although the injection of bacterial cells allows the rapid identification of resistant plants, it is important to note that the results are conclusive only for the isolate UFV32, which was chosen because of its aggressiveness observed in previous experiments (data not shown). If a strain of an unknown level of virulence is used in screening for resistance of Eucalyptus spp. to bacterial wilt, it is likely that results will be different.

Acknowledgments This research was supported by CNPq, FAPEMIG and CMPC Celulose Riograndense. The technical assistance of Pollyane S. Hermenegildo is greatly appreciated. We are also grateful to Norton Borges (CMPC) and Reginaldo Gonçalves Mafia (Fibria) for providing the cutting material used in the inoculation experiments and to Embrapa Hortaliças for providing the strain used in this study. Additionally, we are thankful to Fibria and Clonar Resistência a Doenças Florestais, involved in the propagation of plant material used in the inoculation experiments.

References Alfenas AC, Mafia RG, Sartório RC, Binoti DHB, Silva RR, Lau D, Vanetti CA (2006) Ralstonia solanacearum em viveiros clonais de eucalipto no Brasil. Fitopatol Bras 31:357–366 Alfenas AC, Zauza EAV, Mafia RG, Assis TF (2009) Clonagem e doenças do eucalipto, 2ªth edn. Editora UFV, Viçosa Ciesla WM, Diekmann M, Putter CA (1996) Eucalyptus spp. FAO/IPGRI Technical Guidelines for the Safe Movement of Germplasm, n. 17. FAO/IPGRI, Rome Coutinho TA, Roux J, Riedel K-H, Terblanche J, Wingfield MJ (2000) First report of bacterial wilt caused by Ralstonia solanacearum on eucalypts in South Africa. For Pathol 30:205–210 Cruz AP, Dianese JC (1986) Tolerância à murcha bacteriana em eucalipto. Fitopatol Bras 11:396 Dianese JC, Dristig MCG (1993) Screening Eucalyptus selections for resistance to bacterial wilt caused by Pseudomonas solanacearum

Trop. plant pathol. In: Hartman GL, Hayward AC (eds) Bacterial Wilt. Proceedings of an International Conference. ACIAR, Kaoshiung. p. 206–210 Dianese JC, Dristig MCG, Cruz AP (1990) Susceptibility to wilt associated with Pseudomonas solanacearum among six species of Eucalyptus growing in equatorial Brazil. Australas Plant Pathol 19:71–76 Fonseca SM, Resende MDV, Alfenas AC, Guimarães LMS, Assis TF, Grattapaglia D (2010) Manual prático de melhoramento genético de eucalipto. Editora UFV, Viçosa Fonseca NR, Guimarães LMS, Hermenegildo PS, Teixeira RU, Lopes CA, Alfenas AC (2014) Molecular characterization of Ralstonia solanacearum infecting Eucalyptus spp. in Brazil. For Pathol 44: 107–116 Kado EI, Heskett MG (1970) Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60:969–976 Kelman A (1954) The relationship of pathogenicity in Ralstonia solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693 Lopes CA, Boiteux LS (2012) Melhoramento para Resistência a Doenças Bacterianas. In: Fritsche-Neto R, Borém A (eds) Melhoramento de Plantas para Condições de Estresses Bióticos. Suprema Gráfica e Editora, Visconde de Rio Branco, pp 61–88 Mafia RG, Alfenas AC, Penchel Filho RM, Ferreira MA, Alfenas RF (2012) Murcha-bacteriana: disseminação do patógeno e efeitos da doença sobre a clonagem do eucalipto. Rev Árvore 36:593–602 Mafia RG, Alfenas AC, Ferreira MA (2014) Avaliação da resistência do eucalipto à murcha-bacteriana causada por Ralstonia solanacearum. Rev Árvore 38:649–656

Pongpanich K (2000) Eucalyptus pathology in Tailand. In: Eucalyptus diseases and their management, Final Report. ACIAR, Bangkok. p. 6–8 Roux J, Coutinho TA, Wingfield MJ, Byabashaija DM (2001) Diseases of plantation Eucalyptus in Uganda. S Afr J Sci 97:16–18 Santiago TR, Grabowski C, Mizubuti ESG (2014) First report of bacterial wilt caused by Ralstonia solanacearum on Eucalyptus sp. in Paraguay. New Disease Reports 29:2 Sudo S, Oliveira GHN, Pereira AC (1983) Eucalipto (Eucalyptus sp) e Bracatinga (Mimosa escabrela), novos hospedeiros de Pseudomonas solanacearum E.F. Smith. Fitopatol Bras 8:631 Thu PQ, Old KM, Dudzinski MJ, Gibbs RJ (2000) Results of eucalypts disease surveys in Vietnam. In: Eucalyptus diseases and their management. In: Eucalyptus Diseases and Their Management, Final Report. ACIAR, Bangkok. p. 16–18 Wang WY (1992) Survey of Eucalyptus disease in Taiwan. Bull Taiwan For Res Inst 7:179–194 Wei R-P, Luo Z, Fang B (2014) Clonal variation of eucalypts in susceptibility to bacterial wilt detected by using different inoculation methods. Silvae Genet 63:1–2 Wu QP, Liang ZC (1988) Selection of species and provenance of Eucalyptus for resistance to bacterial wilt. J S China Agric Univ 9: 41–45 Yabuuchi E, Kosako Y, Yano I, Hotta H, Nishiuchi Y (1995) Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Douderoff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. & Ralstonia eutropha (Davis 1969) comb. nov. Microbiol and Immunol 39:897–904