RESOLVING THE STATUS OF MONILINIA spp. IN SOUTH AFRICAN

SUMMARY

Geographical distribution records of pathogens andpests are the basis for phytosanitary decision-making.Monilinia fructicola, one of the three Monilinia speciesresponsible for brown rot of stone fruit, is listed as aregulated pest for South Africa. Many disputes aboutthe justification of this classification have arisen duringthe past years as records have been published regardingthe presence of this pathogen in South Africa. Results ofseveral surveys conducted from 1985 in stone fruit or-chards, revealed that M. laxa is the only causal agent ofbrown rot in South Africa. However, another notifica-tion of non-compliance (interception of regulated pests)was received by the National Plant Protection Organisa-tion of South Africa in 2003 stating that M. fructicolawas detected on consignments of South African Prunusfruits (P. domestica) in the United Kingdom. A detectionsurvey according to inspections and sampling relevantto the biology of Monilinia species were conducted inthe identified orchards at various stages throughout theyear. Molecular techniques with species-specific primersfor M. fructicola, M. laxa and M. fructigena based on theEPPO Diagnostic Protocol for M. fructicola were usedfor the identification of presumptive positive Moniliniaisolates. The absence of M. fructicola from SouthAfrican stone fruit orchards was again confirmed by thissurvey and its status in South Africa can be reported as:absent, not known to occur, confirmed by a detectionsurvey. The regulated status of M. fructicola in SouthAfrica is therefore scientifically justified by the resultsfrom this survey.

Key words: Brown rot, EPPO protocols, detectionsurvey, Monilinia fructicola, Monilinia laxa, Prunus,quarantine.

Corresponding author: E. CarstensFax: +27.21.8828557E-mail: [email protected]

INTRODUCTION

Brown rot is one of the most important pre- and post-harvest fungal diseases known to the fruit industry(Ogawa and English, 1991; Michailides and Morgan,1997). Three species of the fungal genus Monilinia Hon-ey, namely Monilinia fructicola (G. Winter) Honey, M.laxa (Aderh. & Ruhland) Honey and M. fructigena Hon-ey, are the casual organisms of brown rot (Byrde andWillets, 1977; Ogawa et al., 1995; Snyder and Jones,1999). Amongst the three Monilinia species, M. fructicolais considered the most destructive. The three species donot occur in all areas where stone fruit are grown. M.fructicola, known as the American brown rot fungus, iscommon in the Americas (North and South), Australia,New Caledonia and New Zealand (CABI/EPPO, 1999;CPC, 2005), whilst the other two species, indigenous toEurope, are known as the European brown rot fungi(CABI/EPPO, 1991, 2000). All three species are knownto occur together only in Central and Eastern Asia,where Prunus, Malus and Pyrus spp. originated(CABI/EPPO, 1991, 1999, 2000). During 2004, M. fruc-ticola was reported for the first time in China after thepathogen was intercepted in the UK on Prunus fruits im-ported in 2003 (OEPP/EPPO, 2004; Zhu et al., 2005).In Europe, M. fructicola was moved from the A1 pest list(pests not present in the region) of the European andMediterranean Plant Protection Organisation (OEPP/EPPO) to the A2 pest list (pests present in some EPPOcountries and under official control) after detection ofthis pathogen in Austria, France, Spain and the CzechRepublic (OEPP/EPPO, 2002a, 2002b, 2005, 2006a;Duchoslavová et al., 2007; OEPP/EPPO, 2008). In2006, this pathogen was eradicated in Austria (OEPP/EPPO, 2006b).

All three Monilinia spp. cause similar brown rotsymptoms and can be distinguished only by laboratoryexamination. The species are also morphologically simi-lar in culture, and the greatest difficulty is experiencedto distinguish between isolates of M. fructicola and M.laxa (OEPP/EPPO, 1988; EPPO/CABI, 1997; Lane,2002). Several studies have reported species-specificPCR primers for the identification of M. fructicola (Ful-ton and Brown, 1997; Fulton et al., 1999; Snyder and

Journal of Plant Pathology (2010), 92 (1), 35-41 Edizioni ETS Pisa, 2010 35

RESOLVING THE STATUS OF MONILINIA spp.IN SOUTH AFRICAN STONE FRUIT ORCHARDS

E. Carstens1,2, J.M. van Niekerk1, W. Laubscher3 and P.H. Fourie1,2

1 Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Stellenbosch, 7602, South Africa2 Citrus Research International, P.O. Box 2201, Stellenbosch, 7602, South Africa

3 Department of Agriculture, Private Bag X5015, Stellenbosch, 7599, South Africa

003_JPP217RP(Carstens)_35 6-04-2010 17:07 Pagina 35

36 Monilinia spp. on stone fruit in South Africa Journal of Plant Pathology (2010), 92 (1), 35-41

Jones, 1999; Förster and Adaskaveg, 2000; Hughes etal., 2000; Ioos and Frey, 2000; Boehm et al., 2001; Ma etal., 2003; Côte et al., 2004a, 2004b). However, some ofthese methods only differentiate M. fructicola from M.laxa, but have not been validated to distinguish M. fruc-ticola from M. fructigena. According to the EPPOCouncil responsible for the approval of EPPO Stan-dards, Hughes et al. (2000) and Ioos and Frey (2000)developed the most reliable PCR primers. These twoprimer sets were included into the standard approvedby the EPPO council that describes a diagnostic proto-col for M. fructicola, a regulated pest for EPPO(OEPP/EPPO, 2003a).

South Africa is an export-driven country and ex-porters and the stone fruit industry (apricots, nectarines,peaches and plums) are constantly seeking access to newmarkets and expansion of existing markets. South Africais currently the largest producer of apricots and thethird largest producer of stone fruit in the southernhemisphere. The total value of plum production for2006/7 in South Africa was R 271,494,000 (DFPT,2008). From a quarantine perspective, stone fruit is rat-ed as “high risk crops” as a number of devastating exot-ic pests associated with stone fruit such as M. fructicolaare currently absent from South Africa. M. fructicola islisted as a regulated pest for South Africa (Anonymous,2008). For a pathogen to be classified as regulated pestfor a country, the pest should be of economic impor-tance to the endangered area and not yet present there,or present but not widely distributed and being official-ly controlled (FAO, 2002).

Many disputes have, however, arisen during the pastyears concerning this classification due to officialrecords of the presence of this pathogen dating back tothe 1950s. The first reference concerning the presenceof M. fructicola in South Africa was by Doidge et al.(1953). Similar information was published by Heyns(1967) and Gorter (1977) about the presence of thispathogen in the South-Western and Southern Cape andalso sporadic in Transvaal (= Gauteng). The situationwas further complicated by reports from some Euro-pean countries on detection of M. fructicola on stonefruit imported from South Africa (OEPP/EPPO, 1999;Van Leeuwen et al., 2001).

Thus, several surveys were conducted to ascertainwhether this pathogen is present on stone fruit in theSouth-Western and Southern Cape. The results of thesesurveys were based solely on qualitative traits dependingon colony characteristics and confirmed that M. fructico-la was not present in these two stone fruit production ar-eas, and that brown rot in South Africa is caused by M.laxa only (Matthee, 1970; Schlagbauer and Holz, 1987;Den Breeyen, 1994). Based on the outcome of these sur-veys the status of M. fructicola in South Africa waschanged to absent, formerly present (CABI/EPPO,1999). Research done by Fourie et al. (2002) also con-

firmed that brown rot in South Africa is caused by M.laxa. Results from this study were also based on qualita-tive traits depending on colony characteristics.

However, a notification of non-compliance (intercep-tion of regulated pests) was again received by theNPPO of South Africa during April 2003 from the UK,stating that M. fructicola was detected on consignmentsof Prunus fruits (P. domestica) traced back to orchardsin South Africa (OEPP/EPPO, 2003b). The orchardswhere the fruit originated were traced to productionunits located in the Gauteng Province (previouslyknown as the Transvaal), and to fruit of the plum cvsFlavor King, Songold and Leatitia.

The aim of this study was to ascertain whether M.fructicola is present in South African stone fruit or-chards according to the terms of international standardsfor phytosanitary measures (ISPM) and based on diag-nostics molecular protocols that contain adequate re-quirements for a reliable diagnosis of the specificspecies (OEPP/EPPO, 2003a; 2006c). The ISPMs ofparticular relevance are ISPM No. 6, “Guidelines forsurveillance” (FAO, 1997) and ISPM no. 8, “Determi-nation of pest status in an area” (FAO, 1998).

MATERIALS AND METHODS

Detection survey. The survey was conducted withrelevance to the biology of M. fructicola in the identifiedorchards of the three plum cultivars located in theGauteng Province. Three Songold orchards (average of3.43 ha per orchard) and three Leatitia orchards (aver-age of 3.68 ha per orchard) as well as one Flavor Kingorchard of 1.4 ha, with 1000 trees per ha, were inspect-ed and sampled. The Songold and Leatitia orchardswere established in 1997, 1998 and 2000 and the FlavorKing orchard in 1997.

The number of trees per orchard inspected for symp-toms, which included decayed or mummified fruit, blos-som and twig blight and cankers, was determined accord-ing the statistical method of Cannon and Roe (1982). Toprovide for a 95% confidence of sampling, 138 treeswere randomly sampled per 1000 trees per ha.

The first inspection and sampling was at bud burstduring August 2003, and all the orchards were moni-tored for mummified fruit that had fallen to the groundor remained hanging on the trees, as well as for cankerswith profuse gumming. The second inspection andsampling was at full bloom stage during September2003 when all the orchards were monitored for symp-toms of blossom and twig blight. The third inspectionand sampling was during October 2003 when all the or-chards were monitored for twig blight symptoms andyoung fruit symptoms. The final inspection and sam-pling were carried out at later stages of harvesting dur-ing December 2003 and January 2004 with special em-


Journal of Plant Pathology (2010), 92 (1), 35-41 Carstens et al. 37

phasis on fruit expressing brown rot symptoms.

Fungal isolates and colony characteristics. Isolationswere made from the 420 samples. Small pieces of tissue(mummified fruit, twigs and young fruit) were plateddirectly onto malt agar (MA; Biolab, South Africa), po-tato carrot agar (PCA; 20 g Biolab agar, 20 g carrot, 20 gpotato, 1000 ml water) and potato-dextrose agar (PDA;Biolab, South Africa) amended with 2% streptomycin.The plates were incubated at 25°C. Plates were moni-tored daily for presumptive Monilinia isolates based ongeneral colony characteristics. Hyphae growing outfrom the tissue pieces were subcultured onto fresh PDAand water agar (WA; Biolab) to induce sporulation. Iso-lates were incubated at 25°C under near-ultraviolet light(Dhingra and Sinclair, 1995). Presumptive positive iso-lates were hyphal-tipped to obtain pure cultures andmaintained on PDA. Disks of all the cultures (6-day-old) were transferred to oatmeal agar (OMA; Biolab) in

Petri dishes, and placed in a pattern as described bySonoda et al. (1982), to examine the interaction be-tween the different cultures.

DNA isolation and amplification using species-specific primers for the identification of M. fructicola.All the presumptive positive Monilinia isolates weregrown on PDA. Twenty isolates collected from stonefruit during the 1996/97, 1997/8 and 1998/99 seasons(Fourie et al., 2002) were also included. Total genomicDNA was extracted using the Qiagen DNeasy PlantMaxi Kit (Hilden, Germany). The species-specificprimer pair 5’-TATGCTCGCCAGAGGATAATTA-3’(Mfc-F1) and 5’-GATTTTAGAGCCTGCCATTA-3’(Mfc-R1) developed by Hughes et al. (2000) was used.These primers are based on subtle DNA sequence dif-ferences in the ITS 1 and 2 regions of the nuclear rRNAgene repeat. The species-specific primer pairs were cus-tom synthesised by Integrated DNA Technologies(Coralville, USA). Amplification with the primers spe-cific for M. fructicola was performed using the PCR con-ditions recommended by Hughes et al. (2000) as pub-lished in the EPPO-approved diagnostic protocol forM. fructicola (OEPP/EPPO, 2003a), except for the an-nealing temperature, which was increased from 59°Cfor 1 min to 62.5°C for 30 sec. Positive controls (DNA

Fig. 1. Gel documentation of PCR products following PCRamplification of DNA extracted from Monilinia cultures ex‘Leatitia’ plum with species-specific primers for M. fructicola.Samples on the gel are as follows: lane 1 denotes a 100-bpDNA ladder; lanes 2-16, isolates from ‘Leatitia’ plum. Isolatesin lanes 17-19, lane 20 and lane 21 are M. fructicola, M. laxaand M. fructigena positive controls, respectively; lane 22 de-notes a negative water control; lane 25 denotes a 100-bpDNA ladder; lanes 26-40, isolates from ‘Leatitia’ plum. Iso-lates in lanes 41-43, lane 44 and lane 45 are M. fructicola, M.laxa and M. fructigena positive controls, respectively; lane 46denotes a negative water control.

Fig. 2. Gel documentation of PCR products following PCRamplification of DNA extracted from Monilinia cultures ex‘Songold’ plum with species-specific primers for M. fructicola.Samples on the gel are as follows: lane 1 denotes a 100-bpDNA ladder; lanes 2-16, isolates from ‘Songold’ plum. Iso-lates in lanes 17-19, lane 20 and lane 21 are M. fructicola, M.laxa and M. fructigena positive controls, respectively; lane 22denotes a negative water control; lane 23 denotes a 100-bpDNA ladder; lanes 24-38, isolates from ‘Songold’ plum. Iso-lates in lanes 39-41, lane 42 and lane 43 are M. fructicola, M.laxa and M. fructigena positive controls, respectively; lane 44denotes a negative water control.



of M. fructicola, which was obtained from The CentralScience Laboratory, Sand Hutton, York, UK) and nega-tive controls (using water instead of template DNA)were included. DNA of M. fructigena and M. laxa (ob-tained from The Central Science Laboratory, Sand Hut-ton, York, UK) were also included. PCR products wereanalysed by electrophoresis at 100 V for 1 h in a 1%(w/v) agarose gel and visualized under UV light using aGeneGenius Gel Documentation and Analysis System(Syngene, UK) after ethidium bromide staining.

DNA isolation and amplification using species-specific primers for the identification of M. laxa. Thirtyfungal isolates were randomly selected. The species-spe-cific primer pair 5’-TATGCTCGCCAGAGAATAATC-3’(IST1Mlx) and 5’-TGGGTTTTGGCAGAAGCA-CACC-3’ (IST4Mlx) developed by Ioos and Frey (2000)was used in the amplification reaction. These primers arebased on base substitutions between the three Moniliniaspecies clustered in two polymorphic regions, one locat-ed in the ITS1 and the other in the ITS2. The species-specific primer pairs were custom synthesised by Inte-grated DNA Technologies (Coralville, USA). Amplifica-tion with the species-specific primers for M. laxa was per-formed using the PCR conditions recommended by Ioosand Frey (2000) as published in the diagnostic protocolfor M. fructicola, provided by EPPO (OEPP/EPPO,2003a). Positive controls (DNA of M. laxa, obtainedfrom The Central Science Laboratory, Sand Hutton,York, UK) and negative controls (using water instead oftemplate DNA) were included. DNA of M. fructicola andM. fructigena (obtained from The Central Science Labo-ratory, Sand Hutton, York, UK) were also included. PCRproducts were analysed as described above.

RESULTS

Detection survey. A total of 414 trees for each of theSongold and Leatitia orchards and 138 trees for FlavorKing were monitored at each of the four inspection in-tervals. During the surveillance period 98 samples werecollected from the Flavor King orchard, 154 from theLeatitia orchards and 168 from the Songold orchards.Mummified fruit and cankers on twigs were the mostprevalent type of symptoms found in all the identifiedorchards.

Fungal isolates and morphology. Of the 420 samplestaken during the surveillance period, 172 presumptiveMonilinia isolates were obtained through visual exami-nation based on morphological features such as colonymargins, lobing and colour of the sporulating areas. Onthe oatmeal agar plates, no distinct black lines, whichare a positive identification for M. fructicola, were ob-

served between any of these cultures after 10 days incu-bation and the 172 isolates were regarded as M. laxa. Alight line was, however, observed between some of thecultures. Therefore, for conclusive identification, 90 iso-lates (including the aforementioned), 30 each of the dif-ferent cultivars, were selected for molecular analysis.

DNA isolation and amplification using species-specific primers for the identification of M. fructicola.None of the 90 DNA samples of the presumptive posi-tive isolates or DNA from the 20 isolates from Fourie etal. (2002) gave positive results following PCR amplifica-tion with the species-specific primers for M. fructicola.Only DNA from the three M. fructicola positive controlsyielded an amplicon of 280 bp (Fig. 1-4).

DNA isolation and amplification using species-specific primers for the identification of M. laxa. Allthe samples gave positive results following PCR amplifi-cation with the species-specific primers for M. laxa. Allthe isolates, including the positive control (DNA of M.laxa) yielded an amplicon of 350 bp. No amplificationwas observed in the lanes containing the reference DNAof M. fructicola or M. fructigena.

Fig. 3. Gel documentation of PCR products following PCRamplification of DNA extracted from Monilinia cultures ex‘Flavor King’ plum with species-specific primers for M. fructi-cola. Samples on the gel are as follows: lane 1 denotes a 100-bp DNA ladder; lanes 2-16, isolates from ‘Flavor King’ plum.Isolates in lanes 17-19, lane 20 and lane 21 are M. fructicola,M. laxa and M. fructigena positive controls, respectively; lane22 denotes a negative water control; lane 25 denotes a 100-bpDNA ladder; lanes 26-40, isolates from ‘Flavor King’ plum.Isolates in lanes 41-43, lane 44 and lane 45 are M. fructicola,M. laxa and M. fructigena positive controls, respectively; lane46 denotes a negative water control.



DISCUSSION

Distribution records of pests are the basis for phy-tosanitary decision-making (Pasiecznik, et al., 2005). It istherefore imperative for NPPOs to have access to accu-rate information about the geographical distribution ofpests in their own country as well as in other countries. Inorder to obtain this information, accurate identificationof these pathogens or pests is crucial and the assurance ofthe accuracy of this information is very important.Records should preferably be based on results of surveysor positive identifications published in peer-reviewedpublications (Van Halteren, 2000). According to theWTO-SPS, phytosanitary measures have to be scientifi-cally justified and in practice it means that NPPOs musthave access to scientific documentation that they can citeas justification in cases of any possible dispute. This in-formation will also allow for more detailed justification ofthe absence of a pest or pathogen (“not known to occur”to “known not to occur”) in a country (Van Halteren,2000).

In this study, a molecular approach was followed todetermine the presence of M. fructicola in South Africanstone fruit orchards after a thorough detection surveywas conducted. None of the isolates tested with thespecies-specific PCR primers gave positive results forM. fructicola. The PCR test was performed using thePCR conditions of the EPPO-approved and recom-mended diagnostic protocol for M. fructicola(OEPP/EPPO, 2003a), with one exception: annealingtemperature was adjusted to 62.5°C for 30 sec to elimi-nate non-specific amplification, which was observed atthe recommended annealing temperature of 59°C for 1min (results not shown). The absence of M. fructicolawas validated by the positive results following PCR of30 randomly selected isolates with species-specificprimers for M. laxa.

Since 1985, various surveys have been conducted instone fruit orchards in all the production areas (WesternCape and Gauteng), and M. fructicola has never beendetected (Matthee, 1970; Schlagbauer and Holz, 1987;Den Breeyen, 1994; Fourie et al., 2002). This studytherefore confirms these reports, which were based onmorphological identification and provides the scientificjustification to the NPPO of South Africa to supportthe status of M. fructicola as a regulated pest. The statusof M. fructicola in South Africa can therefore be de-scribed as: “Absent, not known to occur, confirmed bya detection survey”.

ACKNOWLEDGEMENTS

This work was financially supported by The Decidu-ous Fruit Producers Trust of South Africa. The authorsthank the South African Department of Agriculture andthe Department of Plant Pathology, University of Stel-lenbosch for technical and administrative support. Theauthors are very grateful to Prof. Pedro Crous for hiscritical review on this manuscript and for his advice onthis research.

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Fig. 4. Gel documentation of PCR products following PCRamplification of DNA extracted from Monilinia cultures exPrunus cultivars (Fourie et al., 2000) with species-specificprimers for M. fructicola. Samples on the gel are as follows:lane 1 denotes a 100-bp DNA ladder; lanes 2-12, isolatesfrom Prunus cultivars. Isolates in lanes 13-15, lane 16 andlane 17 are M. fructicola, M. laxa and M. fructigena positivecontrols, respectively; lane 18 denotes a negative water con-trol; lane 20 denotes a 100-bp DNA ladder; lanes 21-30, iso-lates from Prunus cultivars. Isolates in lanes 31-33, lane 34and lane 35 are M. fructicola, M. laxa and M. fructigena posi-tive controls, respectively; lane 36 denotes a negative watercontrol.



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Received March 26, 2008Accepted August 20, 2009



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RESOLVING THE STATUS OF MONILINIA spp. IN SOUTH AFRICAN