Evaluation of reactions of commercial and autochthonous apple cultivars to common diseases in Serbia under natural infection

SUMMARY This study presents the results of a multiple-year evaluation (1991-1996; 2005-2007) of susceptibility of more than 100 apple cultivars to Venturia inaequalis, Podosphaera leucotricha and Erwinia amylovora under agroecological conditions existing in Serbia. Some of the most popular cultivars were found highly susceptible to V. inaequalis (Cripps Pink, Mutsu, Gloster 69, Wellspur); while Golden Delicious, Richared, Gala, Čačanska pozna, Čadel and Jonagold were susceptible; Idared, Granny Smith and Jonathan moderately susceptible; Lord Lamburne and London Pepping, as well as several autochthonous cultivars were moderately resistant; and a group of resistant cultivars included Prima, Priscilla, Williams Pride, Dayton, Enterprise, Gold Rush, Golden Orange, many of the Re-cultivars (Germany), as well as Baujade, Selena, Dukát, Produkta, Topaz, some older cvs. (Worcester Pearmain, Merton Worcester, James Grieve, Akane, Astilish, Astrachan Red and Discovery), some Co-op selections (USA) and NS hybrids (Serbia). Regarding powdery mildew, Idared and Jonathan were highly susceptible; Gala, Akane, Jonagold, Priscilla, Mutsu, Čačanska pozna, Golden Delicious, Granny Smith, Čadel and GoldRush were susceptible; Wellspur, Astrachan Red, Richared, Jonadel, Dayton and several autochthonous cultivars were moderately susceptible; Lord Lamburne, Astlisch, Prima, Champagne Reinette, Discovery and many autochthonous cultivars were moderately resistant; while most Re-cvs. (Germany), several cultivars from the Czech Republic, some selections from the USA and UK and most NS hybrids (Serbia) were resistant. Also, some cultivars showed variable susceptibility depending on location (Williams Pride, Gloster 69, Baujade and Produkta). E. amylovora was observed only in 2007 and at relatively low intensity (up to 12% infection). The highest disease severity was observed on cv. Elstar, then Granny Smith, Idared and Jonagored; while the lowest was found on Red Chief and Hapke apple trees.


INTRODUCTION
Intensive apple production can ensure high yields and fruit quality, but it also requires high investments, largely in chemical control, which is performed for the purpose of suppressing some economically most significant diseases, such as scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha) and bacterial fire blight (Erwinia amylovora). In some European regions, the number of fungicide treatments against apple diseases exceeds 30, with around 20 to control scab (Evans et al., 2000). In Serbia, the whole program of chemical control for apple trees is focused on scab disease (Balaž & Petrina, 2003). Fungicides account for 30% of total pesticides used in Europe, emphasizing their use in contemporary production systems, such as fruit orchards (Gullino & Kuijpers, 1994). Many European governments presently take measures to reduce the use of pesticides in order to alleviate toxicological and environmental problems. In the late 1970s, European countries accepted the now wellknown concept of Integrated Pest Management (IPM) as the most optimal trend in plant protection. Within that framework, cultivation of resistant apple varieties has a special significance. Fungicide treatments required on a plantation of resistant apple cultivars could be one fourth of those needed to protect susceptible cultivars (Parisi et al., 1995). In the last few decades, several breeding centers have been developed across the world (USA, Canada, France, England, Germany, Switzerland, Czech Republic) in which significant results have been achieved in developing apple cultivars either resistant or with low susceptibility to economically harmful diseases. Using various sources of resistance to scab, powdery mildew and fire blight, as well as conventional and modern molecular methods, a number of cultivars, lines and other selection material have been created. Today, one of the basic aims of selection efforts is to create apple cultivars with high fruit quality, high yield and long-term resistance against the most significant pathogens V. inaequalis, P. leucotricha and E. amylovora.
This paper presents the results of a long term study of the susceptibility/resistance of a variety of introduced and autochthonous apple cultivars and selections to the most common diseases that occur under Serbian agroecological conditions.

MATERIALS AND METHODS
A study of susceptibility of apple cultivars and genotypes to scab and powdery mildew conducted from 1991 to 1996 Apple orchard at Rimski Šančevi (Southern Bačka, Vojvodina). Thirty-seven apple cultivars were studied in the orchard, which was planted in 1989 (Table 1). All cultivars were grafted onto rootstock M9, trained as a slender spindle with a planting distance of 4 x 1.75 m. Multiple-year testing of apple susceptibility to V. inaequalis (1991-1992; 1995-1996) and P. leucotricha (1992)(1993)(1994) was performed. Between 5 and 8 preventive chemical treatments with fungicides such as mancozeb, captan, propineb and sulfur were applied in the orchard during each growing season. First treatment was usually performed with copper fungicides at the growth stage BB CH: 09-10.
Apple orchard at Gladnoš (Srem, Vojvodina). The orchard was planted in 1991. It comprised different genetic material: autochthonous, some standard and some more recent cultivars, wild apples, as well as some selected genotypes, 28 in total (Table 1). The training system was slender spindle on rootstock M26; planting distance 4 x 2 m. The susceptibility of apple cultivars to P. leucotricha was studied over the period 1993-1995, when conventional fungicides were not applied.

A study of susceptibility of apple cultivars and genotypes to scab, powdery mildew and fire blight from 2005 to 2007
Apple orchards at Rimski Šančevi and Kać (Southern Bačka, Vojvodina). Long term experiments were performed at two locations: Rimski Šančevi (fruit growing and viticulture experimental orchards of the Faculty of Agriculture, Novi Sad) and Kać (MAVM commercial nursery). The apple trees planted on these locations are mostly new cultivars with monogenic resistance to V. inaequalis, as well as some autochthonous and standard cultivars, as well as some promising genotypes. The orchards were planted over the period 2000-2002. A total of twenty-three cultivars and some new genotypes and different autochthonous cultivars were studied in the experimental orchards Rimski Šančevi and Kać (Table 1). In both orchards, the susceptibility/ resistance of those cultivars to V. inaequalis and P. leucotricha (2005)(2006) was studied. The training system was slender spindle with rootstock M26, and the planting distance was 4 x 1.5 m. Conventional chemical control was not performed in any of the experimental orchards.
In another field experiment at Rimski Šančevi, several commercial cultivars (Golden Delicious clone B, Golden Delicious clone Reinders, Jonagored, Idared, Granny Smith, Elstar, Red Chief and Hapke) were tested for their susceptibility/resistance to E. amylovora. The training system was slender spindle on rootstock M9 (except for cv. Red Chief grafted onto rootstock M-26) and the planting distance was 4.00 x 1.50 m. Monitoring was performed during 2005-2007. Special chemical control for prevention and suppression of fire blight was not applied, but chemical control of scab and powdery mildew was regularly performed.  1991-1992; 1995-1996 1992-1994 1993-1995 2005-2006   Meteorological data for study periods (1991-1996; 2005-2007), location Novi Sad

Susceptibility evaluation
The susceptibility of apple cultivars to V. inaequalis and P. leucotricha was checked on 3-8 trees representing each cultivar, two-to-three times during each vegetation season (mid-June, early July, and optionally early September). For E. amylovora infection, 100 trees were evaluated.
On the basis of our multiple-year testing of susceptibility/ resistance of various apple cultivars and different genotypes to V. inaequalis and P. leucotricha, the mean value (M) of disease severity was calculated for each cultivar. According to these results, the tested cultivars were classified into specific groups based on their susceptibility or resistance: resistant M=0; moderately resistant M=0.01-2.00; moderately susceptible M=2.01-3.00; susceptible M=3.01-5.00 and highly susceptible: M=5.01-6.00.
Regarding cultivar susceptibility to E. amylovora, only the percentage of disease was evaluated (without their classification into specific groups based on susceptibility/ resistance) because a low severity fire blight infection occurred only during one year (2007).
Apple orchard at Gladnoš (Srem, Vojvodina). Powdery mildew (P. leucotricha) was constantly present and spreading over the study period (1993)(1994)(1995), which was expectable because fungicides were not applied. According to the results, the susceptibility of the examined cultivars to P. leucotricha varied in that orchard. The infection level was generally not very high (Figure 4) because the studied cultivars and some genotypes possess genetic resistance.   Table 3).

Study of apple susceptibility to scab, powdery mildew and fire blight in 2005-2007
Apple orchards at Rimski Šančevi (orchards 1 and 2) and Kać (Southern Bačka, Vojvodina). The results of a two-year study (2005)(2006) showed that some newer promising apple cultivars and different genotypes created in the most prominent breeding centers worldwide, expressed resistance to the most harmful diseases (scab and powdery mildew) under our agro-ecological conditions. Some autochthonous cultivars included in this study also showed a high level of resistance, too ( Figures 5 and 6, Tables 2 and 3).
The same cultivars also showed a high level of resistance to powdery mildew (P.  Symptoms of powdery mildew were detected only at very low severity in the cultivars Baujade (France), Produkta (Czech Republic) and only in one Serbian genotype (NS 11/111), and they were grouped as moderately resistant (M=0.01-2.00). Two American cultivars (Dayton and William's Pride) were grouped as moderately susceptible (M=2.01-3.00) ( Table 3).
In the same location (Kać), symptoms of powdery mildew (P. leucotricha) were not detected in most of the examined cultivars (Regine, Remo, Rewena, Renora, Reka, Baujade, Co-op 33, Co-op 34, Co-op 36, Co-op 37, Golden Supreme, Jonagold Wilmuta, Golden Delicious clone Reinders and Topaz). They only appeared in traces on several cultivars (Enterprise, Orahovača, Golden Orange, Cripps Pink, Reanda, Rebela, Remura and Relinda). Such very low occurrence of powdery mildew in that orchard could possibly be attributed to a small amount of primary inoculum due to its more resistant apple assortment and isolation of that orchard from other fruit trees. Strong severity of powdery mildew occurred only in cv. GoldRush (M≈4.00).

DISCUSSION
Scab (V. inaequalis) occurs on a regular annual basis wherever apple trees are grown in Serbia. Disease severity mostly depends on weather conditions (Visser et al., 1974;Sestraş, 2003a), cultivar susceptibility and quality of chemical control. Scab could be successfully suppressed by Integrated Pest Management (IPM), i.e. by creating and introducing new apple cultivars which ensure high yields, fruit quality and resistance to the most damaging diseases (Ciglar, 1998;Ciancio & Mukerji, 2008;Deguine et al., 2009). However, introduction of scab resistant cultivars has not been so successful because of a lack of fruit quality and broken scab resistance in some cultivars.
The cultivars Prima, Priscilla and other cultivars with genetic resistance (V f gene from Malus floribunda 821, named as Rvi6 according to new nomenclature given by Bus et al., 2011) are generally known as highly resistant to V. inaequalis (Cumminis, 2005;Beckerman, 2006;Ames, 2011). According to our long-term observation, race 6 never appeared under the production conditions of Vojvodina.
The resistance of other cultivars from this group, such as Akane, Discovery, James Grieve and Worcester Pearmain is mainly based on polygenic resistance (Fischer & Fischer, 2002;Sandskär, 2003;Quamme et al., 2003). All Recultivars created in the Institute for Fruit Research at Dresden-Pillnitz (Germany) are known as having a high degree of resistance, i.e. triple and multiple resistance (Fischer & Fischer, 2004). Our experiments confirmed that under Serbia's agro-ecological conditions resistance has not been overcome in any of the Germany cultivars carrying the V f (Regine, Remo, Rewena, Renora and Reanda), V A (Reglindis) or Vr gene (Remura).
The resistance of apple cultivars can be conditioned by monogenic or polygenic inheritance. Monogenetic inheritance is based on major genes (e.g. V f gene in cvs. Prima and Priscila). This resistance is high (vertical) and easily transferrable by conventional crossing methods, but this type of resistance is risky because it can be easily overcome by appearance of new races of pathogens. x zumi calocarpa, M. toringo 852 and Old European cultivars (Lespinasse, 1994;Crosby et al., 1992;Bénaouf & Parisi, 2000;Hemmat et al., 2002;Sandskär, 2003). The V f gene from M. floribunda clone 821 as a source of monogenic resistance to V. inaequalis is the most frequently used gene in apple breeding programs. This gene determines high resistance (immunity). Prima is the first cultivar (1970) from a series of PRI apple cultivars (with the V f gene) that have an acceptable fruit size and quality (Gessler & Pertot, 2012). During the period 1970-1990, 48 scab resistant cultivars were created worldwide, of which 37 cultivars contained the V f gene from M. floribunda 821 (Crosby et al., 1992). Scab symptoms on leaves of the cultivar Prima (V f ), as well as on other apple cultivars and selections (originating from M. floribunda 821, and with the progenitor M. floribunda 821 remaining resistant) were noted in 1984 (Germany). The occurrence of scab symptoms on selection material originating from M. floribunda 821 (V f gene) meant that a new race of V. inaequalis appeared which overcame the resistance conditioned by the V f gene. That new race of V. inaequalis was named as race 6, and it was described by Parisi et al. (1993). After only a year, race 7 was described in England (Roberts & Crute, 1994), which could infect all progenies of M. floribunda 821. Five races of V. inaequalis had been described in the earlier period: races 1-3 (Shay & Williams, 1956), race 4 (Williams & Kuć, 1969), race 5 (Williams & Brown, 1968).
Another type of inheritance is polygenic, conditioned by a number of genes. Polygenic resistance is not high (horizontal) and it depends on ecological factors, but it is more durable. The best way of preserving durable resistance is a combination of various sources of resistance. Such genetic base provides the best barrier against infection with pathogens (Evans et al., 2000). Polygenic resistance is usually a characteristic of older traditional apple cultivars, manifesting itself as field resistance to a certain degree.
Our data on P. leucotricha presence showed that cvs. Jonathan and Idared were highly susceptible to powdery mildew, confirming earlier findings (Vojvodić & Vrabl, 1984;Marine et al., 2010;Yoder & Biggs, 2011a). Many well-known cultivars, such as Gala, Akane, Jonagold, Priscilla, Mutsu, Čačanska pozna, Golden Delicious, Granny Smith, Čadel and Gloster 69, were found susceptible. According to literature sources, Golden Delicious, Mutsu, Summerred and Jonagold are susceptible (Yoder & Biggs, 2011a) but Granny Smith was reported as highly susceptible by several authors (Marine et al., 2010;Yoder & Biggs, 2011a). In our experiments, cvs. Priscilla and Williams Pride were susceptible to powdery mildew, but these cultivars are cited in literature as resistant to moderately resistant (Cline et al., 1998;Yoder & Biggs, 2011a;Beckerman, 2006). Apart from that, Williams Pride and Gloster 69 growing on the location Gladnoš were rated as susceptible, while being moderately susceptible on the location Rimski Šančevi. Such inconsistent reaction could be attributed to a difference in the quantity of primary inoculum and variation in climatic conditions on the observed locations. Similarly, cvs. Baujade and Produkta were moderately susceptible on the location Gladnoš, and moderately resistant at Kać. Many of the well-known cultivars, such as Worcester Pearmain, James Grieve, Wellspur, Richared, Jonadel and Dayton, were classified as moderately susceptible cultivars. In literature, most of these cultivars have been described as low susceptible to powdery mildew (Sestraş, 2003b;Beckerman, 2006;Ames, 2011). In the category of moderately resistant cultivars were: Discovery, Champagne Reinette, Prima, Astilish and Lord Lambourne. The cultivar Prima has been reported as resistant to powdery mildew by many authors (Cline et al., 1998;Beckerman, 2006;Yoder & Biggs, 2011a). The cultivars Discovery, Champagne Reinette, Prima, Astlish, Astrachan Red, James Grieve and Worcester Pearmain have already been reported as low susceptible, and suitable for integrated production (Keserović et al., 1993). All autochthonous cultivars showed a relative low susceptibility to powdery mildew. On the location Gladnoš, cvs. Jadrolika, Bobovec, Krivocepelj and Akpetrič were moderately susceptible, and Budimka, Pramenka, Mušanika, Melresa, Kožara, Tetanka, Kolačara, Kumovača, Lipovača and Prancia were moderately resistant. On the location Kać, some of these cultivars (Bobovec, Krivopecelj, Kožara and Budimka) showed resistant reactions, indicating that the quantity of primary inoculum here also had an influence on the severity of powdery mildew disease. A previous study reported by Balaž et al. (1996) indicated that some autochthonous Serbian cultivars (Prancia, Lipovača and Kumovača) were highly resistant to powdery mildew. All of the Re-cultivars (Germany) showed resistant reactions on the location Rimski Šančevi. In some of them, powdery mildew appeared on the location Kać, but only at very low severity (in traces). Fischer and Fischer (2004) reported that cvs. Remo and Rewena were resistant, and Reanda, Regine, Renora and Reglindis low resistant to powdery mildew. The cultivars created in other breeding centres, such as Selena, Dukát, Topaz (Czech Republic), most NS hybrids (Serbia) and some Co-op selections (USA) have also been found resistant. The NS hybrids are the result of multiple-year selection work in Serbia (Ognjanov et al., 2011). The donors of resistance for these hybrids were the leading commercial and autochthonous apple cultivars. On the location Kać (2005Kać ( -2006, powdery mildew appeared with stronger severity only in cv. GoldRush (M≈4.00). In literature sources, this cultivar varies from being classified as resistant (Cumminis, 2005;Ames, 2011) to moderately resistant (Cline et al., 1998), and to susceptible (Beckerman, 2006) or high susceptible (Biggs et al., 2010).
E. amylovora, monitored in the commercial apple orchard at Rimski Šančevi from 2005 to 2007 occurred only in 2007 when weather in the spring was rainy and favourable for infection. Disease appeared only as "shoot blight" and not in a strong severity form (up to 12% of crown infection). The highest infection was noted on the cultivar Elstar, then Idared and Granny Smith; low disease severity was observed on Jonagored and Golden Delicious and the lowest (in traces) on Hapke and Red Chief. A relatively higher disease severity was expected in Idared and Granny Smith because literature references describe them as the most susceptible cultivars to fire blight (Sobiczewski et al., 1997;Beckerman, 2006;Arsenijević & Gavrilović, 2007;Yoder & Biggs, 2011b). In a fire blight epidemic that occurred in Vojvodina in 2000, the highest disease severity was found on cvs. Idared and Gloster 69; medium severity on Granny Smith and Golden Delicious, and the lowest on Prima, Priscilla and Wellspur (Balaž, 2000). These data were supported by a later study (Aćimović et al., 2014), in which symptoms of fire blight on cv. Idared in Vojvodina province were significantly more severe than they were on Golden Delicious. In literature, Idared, Granny Smith and Golden Delicious have been reported as highly susceptible and/or susceptible cultivars; Prima and Priscilla as moderately resistant (Steiner, 2005); and cv. Elstar as moderately/highly susceptible (Koski & Jacobi, 2009) or moderately susceptible (Panić & Arsenijević, 1996;Sobiczewski et al., 1997). Red Chief was moderately resistant, which is consistent with other reports (Koski & Jacobi, 2009).