S_H1 leaf rust and bacterial halo blight coffee resistances are genetically independent

ABSTRACT

Coffee resistance to Pseudomonas syringae pv. garcae has been associated to pleiotropic effect of S_H1 allele, present in coffee plants resistant to certain races of Hemileia vastatrix, the causal agent of leaf rust, or genetic linkage between resistance alleles to both pathogens. To validate this hypothesis, 63 coffee plants in F₂ generation were evaluated for resistance to 2 isolates of H. vastatrix carriers of alleles, respectively, _v2, _v5 (isolate I/2015) and _v1; _v2; _v5 (isolate II/2015) with the objective to confirm presence of S_H1 allele in resistant plants to isolate I/2015. The same coffee plants were evaluated for resistance to a mixture of P. syringae pv. garcae strains highly pathogenic to coffee. Results showed that, among F₂ coffee allele S_H1 carriers, resistant to isolate I/2015, resistant and susceptible plants to bacterial halo blight were found; the same segregation occurs between F₂ homozygous for S_H1 allele, susceptible to the same isolate (I/2015) of H. vastatrix. Results also indicate that there is no pleiotropic effect of gene or allele S_H1 connection between genes conferring resistance to leaf rust caused by H. vastatrix and bacterial halo blight caused by P. syringae pv. garcae.

Key words
Coffea arabica L.; resistant cultivars; Pseudomonas syringae pv. garcae; linkage; Hemileia vastatrix; pleiotropic effect

INTRODUCTION

Leaf rust is the most important disease of coffee plantations, and widespread in major Coffee arabica L. producing countries. Variability of the causal agent, fungus Hemileia vastatrix Berkeley & Broome, is quite wide, and there are currently 45 known races of this fungus in world (Várzea and Marques 2005). In Brazilian coffee plantations, 17 races have already been identified (Zambolim et al. 2005).

Races of H. vastatrix are characterized by variable combinations of resistance genes, in total of 9, denominated S_H1 to S_H9 (Várzea et al. 2005), with direct implications for pathogenicity and in determining amplitude and nature of their hosts.

Studies conducted by Moraes et al. (1975) with diverse Coffea spp. germplasm exhibiting resistance to different races of H. vastatrix resulted in the identification of sources of resistance to bacterial halo blight, caused by Pseudomonas syringae pv. garcae, in C. arabica exotic varieties of Ethiopian origin, known as Harar, Dilla and Alghe, S 12 Kaffa, and Geisha, all of which are carriers of S_H1 allele.

Multiple resistance to leaf rust and bacterial halo blight presented in these exotic varieties, could be explained according to Carvalho (1988), by the genetic linkage between resistance alleles to both pathogens or pleiotropic effect of S_H1 allele. Sera (2001) and Fazuoli et al. (2009) also attributed to S_H1 allele, present in the Ethiopian varieties (Bettencourt and Carvalho 1968), evaluated by Moraes et al. (1975), the simultaneous resistance to some races of H. vastatrix and bacterial halo blight caused by P. syringae pv. garcae.

According to Bettencourt and Carvalho (1968), S_H1 allele is widespread in major coffee growing areas of Ethiopia. Studies conducted by these authors, allowed S_H1 allele identification in various selections as Barbuk Sudan, BE-2 Ghembi, BE-4 Ennarea, BE-5 Wush-Wush, BE-6 Moderalo, BE-7 Boggia, BE-8 Era, BE-14 Loulo, Dilla & Alghe, Geisha, Lejeune’s, S 6 Cioiccie, S 9 Arba Gougou, S 12 Kaffa, S 17 Yrgalem, U l Dalecho, occurring individually or in combination with other resistance genes, as S_H4 (S_H1 S_H4); S_H5 (S_H1 S_H5) and S_H4 and S_H5 (S_H1 S_H4 S_H5) and conferring resistance to 16 of 24 H. vastatrix races known at the period in which the research was conducted.

With S_H1 allele conferring resistance to both pathogens, the search for sources of coffee resistant plants to P. syringae pv. garcae will be facilitated and occur simultaneous with tests to evaluate resistance to H. vastatrix. Therefore, the present study aimed to investigate whether coffee simultaneous resistance to H. vastatrix and P. syringae pv. garcae is due to pleiotrophic effect of S_H1 allele or genetic linkage between resistance alleles to both pathogens.

MATERIAL AND METHODS

F₂ progenies of coffee, from H8089-2 and H8089-7, composed respectively of 34 and 29 plants, were evaluated to infection by bacterium P. syringae pv. garcae and fungus H. vastatrix. These plants were derived from crossing between Catuaí Vermelho IAC 24 cultivar and IAC 1137-5 of Geisha originally from Ethiopia, carrier of S_H1 and S_H5 alleles (Bettencourt and Carvalho 1968). The Geisha selection was introduced in the coffee collection of Agronomic Institute (IAC) in 1953, coming from the United States Department of Agriculture under registration PI 205.928.

Plants were inoculated with bacteria obtained from the IBSBF Culture Collection of the Biological Institute, Campinas, Brazil, strains IBSBF 75 and IBSBF 1197 of P. syringae pv. garcae, in mixture, with approximate concentration of 10⁸ UFC∙mL⁻¹ (600 nm, absorbance = 0.25), by abrasion technique, which constitutes of friction against ab axial surface of leaves with medium grit sandpaper disposed in circular area (Ø1.5 cm) previously soaked in bacterial suspension causing limb injuries with no tissue drilling. Analysis of disease severity was carried out by using a rating scale of 0 to 5 points adapted from Paradela et al. (1974), as follows: 0 = no anasarca or chlorosis symptoms or hypersensitivity reaction around injured tissues; 1 = initial of bacterial colonization around lesions, with up to 10% of inoculated area showing disease symptom; 2 = 11 – 25% of inoculated area showing disease symptoms and with or without yellow halo; 3 = 26 – 50% of inoculated area showing disease development, yellowish halo pronounced around lesions; 4 = 51 – 75% of inoculated area with necrosis, yellowish halo throughout inoculated area; 5 = 100% necrosis of inoculated area. Plants were evaluated weekly up to 42 days after inoculation.

To confirm the presence of dominant S_H1 allele, 63 F₂ coffee plants were inoculated with 2 isolates of H. vastatrix obtained on differentiating plants grown in Centro de Café Alcides Carvalho from IAC.

The isolates used in this research were I/2015 carrying alleles _V2 and _V5 and isolate II/2015 the carrier of the alleles of virulence _V1; _V2; and _V5. These aspects were confirmed by inoculation of this isolates on known differentiating plants to H. vastatrix races.

Disks of coffee leaves were inoculated with a drop of uredospore suspension with approximate concentration of 1.5 × 10⁵ uredospore∙mL⁻¹ of isolates I/2015 and II/2015, and subsequently kept in humid chambers in accordance with the methodology proposed by Eskes and Toma-Braghini (1981).

Two parameters were used to determine rust reaction: disease severity, evaluated with a 0 to 9 points scale, 0 being assigned to resistant plants without symptoms and 9, plants with high incidence of large lesions and regular and intense sporulation; and also type of reaction of lesions, evaluated by 0 to 4 point scale, in which 0 was assigned to immune plants without injuries and, four points to high susceptible plants with intense sporulation (Eskes and Toma-Braghini 1981).

Possibility of pleiotropic effect of S_H1 allele or any genetic connection between resistance alleles to the pathogens used in this was investigated through reactions of plants to infection by bacteria strains IBSBF 75 and IBSBF 1197 and isolate I/2015 of fungus, and concordance between evaluated methods through following parameters:

• Accuracy of method (AM) = (a + d)/n, calculated from sum of simultaneously resistant plants (a) and simultaneously susceptible (d) to rust and bacterial halo blight divided by total number of plants (n).

• False positive rate (FPR) = b/(b + d), calculated by dividing the number of resistant plants to leaf rust, but susceptible to bacterial halo blight (b) by total of susceptible plants to bacterial halo blight (b + d).

• False negative rate (FNR) = c/(a + c), calculated by division of number of susceptible plants to leaf rust, but resistant to bacterial halo blight (c) by total of resistant plants to bacterial halo blight (a + c).

• Total rate error (TRE) = (b + c)/n, calculated from sum of resistant plants to leaf rust, but susceptible to bacterial halo blight (b) and number of susceptible plants to leaf rust, but resistant to bacterial halo blight (c) and divided by total number of plants (n).

• McNemar chi-square with Yates continuity correction: (χ²_McNemar ) = (|b − c| − 0.5)²/b + c, used to estimate significance between 2 assessments.

• P value = quantitative measure of significance robustness of χ²_McNemar test calculated.

• Yule association coefficient (Q): measure degree of association between determined classes on evaluated plants for resistance to leaf rust and bacterial halo blight.

RESULTS AND DISCUSSION

The results related to resistance expression in progenies F₂ from H8089-2 and H8089-7 to infection by P. syringae pv. garcae and isolates I/2015 and II/2015 of H. vastatrix are shown in Table 1.

Table 1
Reaction to infection by Pseudomonas syringae pv. garcae and isolates I/2015 and II/2015 of Hemileia vastatrix on F₂ progeny from H8089-2 and H8089-7, hybrids between Catuaí Vermelho IAC 24 cultivar and Geisha IAC 1137-5 selection.

Parameter		H8089-2		H8089-7		Total
Plants (n°)		34		29		63
PHV I I/20151 (R:S)		6:28		10:19		16:47
PHV I I/20152 (R:S)		0:34		0:29		0:63
PPSG3 (R:S)		0:34		15:14		15:48

• ¹
  Proportion of resistant and susceptible plants to isolate I/2015 of H. vastatrix;
• ²
  Proportion of resistant and susceptible plants to isolate II/2015 of H. vastatrix;
• ³
  Proportion of resistant and susceptible plants to P. syringae pv. garcae. P = Proportion; R = Resistant; S = Susceptible.

According to the obtained results, F₂ plants are segregating for resistance to isolate I/2015 of H. vastatrix, but they proved to be susceptible to isolate II/2015. On the other hand, 34 plants of H8089-2 were susceptible to bacterial halo blight while 29 progenies of H8089-7 segregate in approximate 1R:1S ratio (Table 1).

From the total progeny analyzed regarding response to infection with isolate I/2015 of H. vastatrix, 16 plants presented resistance reaction and 47 were susceptible. On the other hand, regarding reaction to infection by P. syringae pv. garcae, 15 plants were identified with resistance reaction while remaining 48 showed to be susceptible (Table 1).

These results show heterozygous nature of F₁ matrices (S_H1 s_H1) and, as demonstrated by Bettencourt and Carvalho (1968), genitors, Catuaí Vermelho IAC 24 cultivar and Geisha IAC 1137-5 selection were respectively homozygous for s_H1 and S_H1 alleles. Furthermore, according to these results, it was possible to confirm the presence of dominant allele S_H1 in a third of plants, six coffee progenies of H8089-2 and ten coffee plants of H8089-7. In the gene-to-gene theory (Flor 1971), these coffee plants, carriers of at least one dominant allele S_H1, had broken down resistance when inoculated with isolate II/2015 that carried _v1 allele.

In the hypothesis of S_H1 allele present pleiotropic effect or occurrence of genetic linkage between alleles that confer resistance to H. vastatrix and P. syringae pv. garcae as reported by Carvalho (1988), coffee plants carrying S_H1 allele should be resistant to both pathogens.

Thus, F₂ progenies, segregating, when inoculated with isolate I/2015 of H. vastatrix, should have the same reaction when inoculated with bacterium P. syringae pv. garcae, that is, resistant and susceptible plants to isolate I/2015 of H. vastatrix should be, respectively, resistant and susceptible to P. syringae pv. garcae.

In Table 2, it is presented the comparative data analysis of F₂ progenies reaction with regarding to infections by P. syringae pv. garcae and by isolate I/2015 of H. vastatrix.

Table 2
Total number of F₂ coffee plants of 2 hybrids H8089 resistant and susceptible to Pseudomonas syringae pv. garcae and isolate I/2015 of Hemileia vastatrix.

H. vastatrix isolate I/2015		P. syringae pv. garcae				Total
		Resistant		Susceptible
		n°
Resistant		6a		10b		16a + b
Susceptible		9c		38d		47c + d
Total		15a + c		48b + d		63n

From 63 F₂ coffee plants evaluated, 44 revealed simultaneously resistance (6) and susceptibility (38) to the isolate I/2015 of H. vastarix and to bacterial halo blight. Nineteen plants showed an opposite reaction to biotic agents, 10 of them were resistant to leaf rust and susceptible to bacterial halo blight, and 9 were resistant to P. syringae pv. garcae and susceptible to H. vastarix (Table 2). The results of statistical analysis performed with experimental data are described in Table 3.

Table 3
Estimation of parameters related to coffee F₂ progeny of 2 hybrids H8089 response to infection by the bacterium Pseudomonas syringae pv. garcae and fungus Hemileia vastatrix.

Parameters		H8089-2		H8089-7		Total
Accuracy of the method (%)		82.35		55.17		69.84
False positive rate (%)		17.64		13.79		15.87
False negative rate (%)		0		31.03		14.29
Total rate error (%)		17.64		44.82		30.16
χ2 McNemar		4.17*		1.23ns		0.56ns
P-value		0.04		0.27		0.45
Yule association coefficient		-		0.25		0.43
Standard deviation of Yule association coefficient		-		0.37		0.32
Confidence interval of 95% of Yule association coefficient		-		0.47 - 0.98		0.40 - 1.06

• ^*
  Significant at 5% probability;
• ^ns
  Non-significant; -Non-calculated values, since false negative rate is 0.

Accuracy of method (AM) calculated from comparative data analysis of coffee reaction to infection by P. syringae pv. garcae and isolate I/2015 of H. vastatrix denote that plants simultaneously resistant or susceptible to both pathogens was 69.84% considering total number of F₂plants in the same group the total rate error (TRE) was equal to 30.16%.

The χ²_McNemar test was significant in progenies from H8089-2 (4.17; p = 0.04), not significant in progenies from H8089-7 (1.23; p = 0.27) and also not significant in total group of plants (0.56; p = 0.45). However, significant value of χ²_McNemar test calculated on H8089-2 progeny reveals inconsistency between the 2 classifications; the number of resistant plants to isolate I/2015 of H. vastatrix (6) is not the same number of resistant plants to P. syringae pv. garcae (0).

On the other hand, even though χ²_McNemar value was not significant, in the analysis of all plants evaluated, resistance to biotic agents was not proven to be simultaneous, since 10 plants resistant to the isolate I/2015 of H. vastatrix carrying S_H1 allele proved to be susceptible to bacterial halo blight and 9 plants resistant to bacterial halo blight have shown susceptibility to the isolate I/2015 of H. vastatrix, and false positive rate, in same group of plants, was equal to 15.87% and the false negative in second was 14.29%; the total error rate was 30.16%.

Yule association coefficient (Q) was equal to 0.25 for H8089-7 progeny, 0.40 in total group of plants, and has not been calculated for H8089-2 progeny, since false negative rate, i.e. of susceptible plants to isolate I/2015 of H. vastatrix, but resistant to P. syringae pv. garcae, was equal to 0. The magnitude of Yule coefficient, which ranges between 0 and ±1, observed individually in progeny H8089-7, and on F₂ population as a whole (H8089-2 and H8089-7), confirms the lack of association between resistant and susceptible classes determined in the evaluation ratings of plants resistance to leaf rust and bacterial halo blight.

CONCLUSION

In accordance with the analyzed data, we can conclude that S_H1 gene has no pleiotropic effect or genetic linkage between genes which confer resistance to isolates homozygous for _V1 allele of leaf rust caused by H. vastatrix and to bacterial halo blight caused by P. syringae pv. garcae.

Search for resistance sources to bacterial halo blight for use in breeding programs aiming to develop cultivars with simultaneous resistance to both biotic agents should not be restricted to Coffea germplasm carrying S_H1 gene with resistance to races or isolates of H. vastatrix.

ACKNOWLEDGEMENTS

The authors are grateful to the National Council for Scientific and Technological Development (CNPq) for financial support (Project 479.589/2013-50) and research scholarship to OGF; Coordination for the Improvement of Higher Education Personnel (CAPES) (to LMRR) and Coffee Research Consortium (to MTB), as well as to Dr. Suzete A. Lanza Destéfano, Curator of IBSBF Culture Collection, for providing bacterial strains used in this study, and Dr. Júlio Rodrigues Neto, Dr. Flávia Rodrigues Alves Patrício, Msc. Irene Maria Gatti de Almeida and Dr. Luis Otávio Saggion Beriam for their helpful suggestions.

REFERENCES

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Publication Dates

• Publication in this collection
  15 May 2017
• Date of issue
  Apr-Jun 2017

History

• Received
  09 May 2016
• Accepted
  25 Aug 2016