Phytophthora Root and Crown Rot on Apples in Bulgaria

Phytophthora is a genus of Oomycota responsible for some of the most serious diseases with great economic impact (Judelson and Blanco, 2005). While 54 species were found in the 20th century (Erwin and Ribeiro, 1996) another 51-54 new species have been identified (Brasier, 2008) since the year 2000. They are spread worldwide and have broad range of host plants – fruit trees, citrus, forest and park species. Phytophthora can cause serious damages in orchards and nurseries of apples, cherries, etc. In Bulgaria they have been found first on young apples and cherries (1998-1999) in Plovdiv region (Nakova, 2003). Surveys have been done for discovering disease symptoms in Plovdiv and Kjustendil regions. Isolates have been obtained from infected plant material (roots and stem bases) applying baiting bioassay (green apples, variety Granny Smith) and/or PARP 10 selective media. Phytophthora strains were identified based on standard morphology methods – types of colonies on PDA, CMA, V 8, type and size of sporangia, oogonia and antheridia, and oospores. Cardial temperatures for their growth were tested on CMA and PDA. For molecular studies, DNA was extracted from mycelium using the DNA extraction kit. DNA was amplified using universal primers ITS 6 and ITS 4. Amplification products concentrations were estimated by comparison with the standard DNA. Sequencing was done at the Scottish Crop Research Institute (SCRI, Dundee, Scotland). Phytophthora root and crown rot symptoms first appear in early spring. Infected trees show bud break delay, have small chlorotic leaves, and branches die all of a sudden. Later symptoms are found in August-September. Leaves of the infected trees show reddish discoloration and drop down. Both symptoms are connected with lesions (wet, necrotic in appearance) at stem bases of the trees. Disease spread was 2-3% in most gardens, only in an apple orchard in Bjaga (Plovdiv region) it was up to 8-10%. Morphologically, the isolates acquired from the apple trees were identified as Phytophthora cactorum, P. citrophthora and P. cryptogea. Cardial temperatures for their growth were tested on CMA and PDA. PCR tests with ITS primers 4 and 6 generated a band at about 800 bP. Consequent sequencing showed that 2 strains, Bg 1/1 and Bg 1⁄2, belong to Phytophthora cryptogea.


SUMMARY
Phytophthora is a genus of Oomycota responsible for some of the most serious diseases with great economic impact (Judelson and Blanco, 2005).While 54 species were found in the 20 th century (Erwin and Ribeiro, 1996) another 51-54 new species have been identified (Brasier, 2008) since the year 2000.They are spread worldwide and have broad range of host plants -fruit trees, citrus, forest and park species.Phytophthora can cause serious damages in orchards and nurseries of apples, cherries, etc.In Bulgaria they have been found first on young apples and cherries (1998)(1999) in Plovdiv region (Nakova, 2003).
Surveys have been done for discovering disease symptoms in Plovdiv and Kjustendil regions.Isolates have been obtained from infected plant material (roots and stem bases) applying baiting bioassay (green apples, variety Granny Smith) and/or PARP 10 selective media.Phytophthora strains were identified based on standard morphology methodstypes of colonies on PDA, CMA, V 8, type and size of sporangia, oogonia and antheridia, and oospores.Cardial temperatures for their growth were tested on CMA and PDA.
For molecular studies, DNA was extracted from mycelium using the DNA extraction kit.DNA was amplified using universal primers ITS 6 and ITS 4. Amplification products concentrations were estimated by comparison with the standard DNA.Sequencing was done at the Scottish Crop Research Institute (SCRI, Dundee, Scotland).
Phytophthora root and crown rot symptoms first appear in early spring.Infected trees show bud break delay, have small chlorotic leaves, and branches die all of a sudden.Later symptoms are found in August-September.Leaves of the infected trees show reddish discoloration and drop down.Both symptoms are connected with lesions (wet, necrotic in appearance) at stem bases of the trees.
Disease spread was 2-3% in most gardens, only in an apple orchard in Bjaga (Plovdiv region) it was up to 8-10%.
Morphologically, the isolates acquired from the apple trees were identified as Phytophthora cactorum, P. citrophthora and P. cryptogea.Cardial temperatures for their growth were tested on CMA and PDA.
PCR tests with ITS primers 4 and 6 generated a band at about 800 bP.Consequent sequencing showed that 2 strains, Bg 1/1 and Bg ½, belong to Phytophthora cryptogea.

INTRODUCTION
Phytophthora is a genus of Oomycota responsible for some of the most serious diseases with great economic impact (Judelson and Blanco, 2005).While 54 species were found in the 20 th century (Erwin and Ribeiro, 1996) another 51-54 new species have been identified after the year 2000 (Brasier, 2008).They are spread worldwide and have broad range of host plants -fruit trees, citrus, forest and park species.Phytophthora can cause serious damage to apples, cherries and other trees in orchards and nurseries (Newhook, 1959;Jeffers and Aldwinckle, 1987;Ellis, 1997;Growe, 1997;Teviotdale and Gubler, 1999;Wilcox, 1990Wilcox, , 1998)).Pear and plum trees appear to be relatively resistant.
Diseases caused by Phytophthora species are known in literature as "Phytophthora root and crown rot".In Bulgaria, symptoms of Phytophthora root, crown and collar rots were first found on young apple and cherry trees in the region of Plovdiv during 1998-1999 (Nakova, 2003).
Trees declining and dying from Phytophthora root and crown rots are frequently misdiagnosed as suffering from "wet feet" (root asphyxiation) and sometimes confused with those suffering from winter injury (Ellis, 1997).
Symptom expression depends upon how much of the root or crown tissues are affected, and how quickly they are destroyed.Crown rots advance rapidly and trees collapse after the first warm weather in spring (Teviotdale and Gubler, 1999).Leaves wilt, dry and remain attached to the tree.Phytophthora infections typically kill young trees.Ellis (1997) states that wet soils that remain saturated for extended periods are required for disease development.Above-ground symptoms vary between tree species but generally include: reduced tree vigor and growth, yellowing or chlorosis of leaves, and eventual collapse or death of the tree.Infected trees may decline slowly over one or more years, or they may collapse in spring.Trees may also appear healthy in spring but die suddenly in the latter part of the growing season.Below-ground symptoms include reddish-brown discoloration of the inner bark and wood.A sharp line demarcates the diseased and healthy portion of the crown.Similar symptoms can be found on the roots (Ellis, 1997;Hickey and Yoder, 2001).
Periods of 24 hrs or more of saturated soil favor Phytophthora infections.Conversely, good soil drainage and more frequent but shorter irrigation reduce the risk of root and crown rot (Teviotdale and Gubler, 1999).
Disease is more often observed in low areas of the orchard on heavy, poorly drained soils (Wilcox, 1998;Hickey and Yoder, 2001).
Phytophthora root and crown rots are caused by several Phytophthora species.All of them require extremely wet soils in order to infect and cause significant damage but they differ in destructiveness depending on plant species (Ellis, 1997).

MATERIAL AND METHODS
Phytophthora fungi causing root and crown rot on apples in Bulgaria were first reported in the period 1998-1999 (Nakova, 2003).Further surveys for discovering disease spread in fruit-growing regions of Bulgaria were done over the period 1999-2009.
Both procedures are standard methods for isolating Phytophthora fungi from infected wood and soils.Plant tissue showing symptoms (from root or crown zones) was thoroughly washed with running water, surface sterilized with alcohol (90%) and cut into small pieces.They were then plated into Petri dishes with PARP media.
Sterilized woody tissues were also used in "baiting bioassay".Green apples served as "trap culture".They were surface sterilized, and then sterile cone-shaped cuts were made.Diseased tissue was placed in the holes, then 4-5 ml of sterile water was added, and wax and parafilm as a cover.The infected fruits were incubated in a growth chamber (25 o C, RH 75% and 12 hrs photoperiod) for 7-10 days until disease symptoms appeared.Fruit tissues from the edge of rotten zones were transferred onto PARP media.
Phytophthora strains were identified based on standard morphology methods: types of colonies on PDA, CMA, V 8, type and size of sporangia, oogonia and antheridia, and oospores.Effects of temperatures on their mycelia growth were tested on CMA, V-8 and PDA in a temperature range from 2 to 35-36 o C.
For molecular studies, DNA was extracted from mycelium using a DNA extraction kit.DNA was amplified using the universal primers ITS 6 and ITS 4 (www.phytophthoradb.org).Amplification products concentrations were estimated by comparison with standard DNA.Sequencing was done at the Scottish Crop Research Institute (M.Cooke, SCRI, Dundee, Scotland).

RESULTS
In Bulgaria, disease symptoms were found first on 2-3-year old apple trees in the region of Bjaga village (Plovdiv) during 1998-1999, and also on 2-year old cherry rootstocks (Katunitza, Plovdiv) and 2-year old cherries (Trilistnik, Plovdiv).Phythophthora root and crown rot was later registered in Kjustendil, Sliven, Jambol, Karnobat, Bourgas and Svichov regions.
Symptoms of Phytophthora root rot disease first appear in early spring.Infected trees show bud break delay, have small chlorotic leaves, and branches die all of a sudden.These symptoms, however, are not enough for a diagnose, and point out that root systems or crowns of the trees can be affected.Reddish brown lesions with wet, necrotic appearance were found on crowns and roots of the infected trees.There is a distinct margin between diseased and healthy wood.Infected wood becomes dark brown and lesions can spread as a ring and also upwards on the trunk of the tree (Figure 1, 2).
Diagnostic symptoms are found also bellow the grafting zone as large dark spots clearly defined from healthy tissues.When bark is removed orange to reddish discoloration shows and a dark line marking the border with healthy tissue.Roots show similar symptoms when infected.
Trees with healthy appearance in the spring can all of a sudden wilt and dry out later in the season (mid-August to September).Leaves on the infected trees drop down early and have reddish discoloration by the end of August.
Studies have pointed out that disease spread is favored by wet, heavy and poorly-drained soils.Heavy rains also provoke disease symptoms.
During the year 1999, a complicated disease syndrome was observed after heavy rainfalls of over 100 l/ m 2 and flooding of young apple gardens aged 2-3 years.In August, when temperatures have reached 30-35 o C at midday, leaves of some trees became reddish or yellowish, started to wilt and entire trees collapsed in a short time.Fruit-growing experts have attributed those symptoms to water and temperature stress.Similar reports, though not always correct, are found also in some publications (Newhook, 1959;Browne and Krober, 1958).Monitoring of some typical fruit growing regions has shown that the disease syndrome has spread some 2-10% and more in some orchards (Table 1).
Spread of the disease varies from 0.63-1% to 8-10%.In an apple garden (Plovdiv, Bjaga) where Phytophthora root and collar rot was first recorded, there was 8-10% of infected trees.Up to 5% was the disease incidence in Kjustendil -the second infection focus.Later, the percentage of diseased trees was between 0.63 and 5%, with one exception of the apple garden in Tzalapitza (close to Plovdiv) -7%.
The isolates obtained in our Laboratory on PARP media or as a result of baiting technique belong mainly to the genus Phytophthora (Table 1).Their pathogenicity was proved by inoculation of young apple fruits and 1-year old apple rootstocks.
Identification of the first 12 isolates obtained from 3-year old apples with symptoms of collar rot (region Plovdiv, village Bjaga) was done on PDA based on fungal morphology and cultural characteristics.Strains isolated from apple trees differ in appearance.The first group on PDA developed whitish, slightly smoky, featherlike colonies with stellate growth (starlike).Sporangiophores are simple or branched, zoosporangia are oval, fussiform or irregular in shape (38-40 x 31-33 mm).Terminal chlamidospores are rarely formed, especially when NH 4 NO 3 was added to the media.Sparse oospores were found (P.cryptogea type).
The second group had whitish, smoky, fluffy mycelia.Mycelia hyphae were long, branched, normal or slightly swollen at the point of branching.Conidiophores were simple or sympodially branched.Zoosporangia were oval to elongated (lemon shape), mostly with papillae, average size 33-34 x 26-28 mm on the media.After a culture period of more than 7 days, terminal or intercalary chlamydospores were found.Antheridia and oogonia, as well oospores, formed in large numbers (P.cactorum type).
The morphology of isolates obtained later from different regions of the country was studied on PDA, V8 and CMA.They showed growth that is typical for Phythophthora: Colonies were stellate or rosette, or patternless on V8 agar.Sporangia were extremely variable in shape -ellipsoid, ovoid, limoniform or irregural.Conidiophores were irregulary branched or in loose sympodium (P.citrophthora type).
Analysing the data from our studies of the morphology of Phytophthora fungi and comparing them with results published by other authors (Smith and Smith, 1906;Smith, 1953Smith, , 1955;;Novotelnova, 1974;Ellis, 1997;Hickey and Yoder, 2001;Erwin and Ribeiro, 1996) we concluded that strains isolated from apples belonged to the species:   Data concerning the effect of temperature on mycelia growth showed that most of Phytophthora's isolates from diseased apple trees developed in the interval of 5-30 o C, the optimum being 20-25 o C. One of the strains started to grow at 2 o C and at 35-36 o C, but slowly on the 7 th day.At high temperatures mycelia were depressed (Tables 2, 3).
PCR tests with Phytophthora isolates from apples were run with ITS primers 4 and 6.All had bands above 700 bP (Figure 6).Two isolates generated a band at about 800 bP.Consequent sequencing of the isolate strains Bg 1/1 and Bg ½ showed that the two strains belonged to Phytophthora cryptogea (Pethybridge and Lafferty) (Figures 7 and 8).

DISCUSSION
In Bulgaria Phytophthora-like type of symptoms were detected first on 2-3-year old apple trees in the region of Bjaga village (Plovdiv) during 1998-1999, and also on 2-year old cherry rootstocks (Katunitza, Plovdiv) and 2-year old cherries (Trilistnik, Plovdiv) (Nakova, 2003).Disease had caused serious damage in apple, cherry and other fruit tree orchards and nurseries.Disease spread- ing varied from 0.63-1.0%to 8-10.0%, calculated based on the percentage of infected trees.Symptoms differ depending on a fruit tree species but generally include: suppressed tree vigor and growth, yellowing or chlorosis of leaves, reddish-brown discoloration of inner bark and wood of the trunk, and finally the collapse or death of the trees (Ellis, 1997;Hickey and Yoder, 2001).
Isolates were obtained from infected plant tissues applying "baiting bioassay" and using PARP 10 media (Erwin and Ribeiro, 1996).Phytophthora strains were identified based on morphology characteristics, cardial temperatures for mycelia growth and molecular tools.Mycelia of most Phytophthora isolates grew in the temperature interval from 5 to 30 o C, with the exception of one isolate that slowly developed at 2 o C and 35-36 o C. High temperatures depressed mycelia growth.PCR tests done on Phytophthora isolates with ITS primers 4 and 6 showed bands above 700 bP.Two isolates generated a band at about 800 bP.Consequent sequencing of those isolates showed that they belonged to Phytophthora cryptogea (Pethybridge and Lafferty).

Figure 1 .
Figure 1.3-year old apples showing symptoms of Phytophthora collar rot -discoloration and wet appearances of inner bark

Figure 2 Figure 3 .
Figure 2. 1-year old apple showing symptoms of Phytophthora collar rot -distinct line between healthy and infected tissues

Table 1 .
Disease spread on apple trees in some regions of Bulgaria

Table 2 .
Influence of temperatures on mycelia growth (in vitro tests) on apple isolate, region Bjaga (Plovdiv), mm

Table 3 .
Influence of temperatures on mycelia growth (in vitro tests) on apple isolate, region Kjustendil, mm