CRI Snykant NAVORSINGSNUUS VAN CITRUS RESEARCH INTERNATIONAL. Julie 2014 Nr 183

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1 CRI Snykant NAVORSINGSNUUS VAN CITRUS RESEARCH INTERNATIONAL Julie 2014 Nr 183 Bepaling van die voorkoms van imazalil weerstandbiedendheid in ʼn sitruspakhuis-omgewing deur Arno Erasmus a, Elaine Basson a, Mareli Kellerman ab, Catherine Savage a, Keith Lesar a, Cheryl Lennox b en Paul Fourie ab a Citrus Research International, Nelspruit b Departement Plantpatologie, Universiteit Stellenbosch Agtergrond. Imazalil (IMZ) is tans ons belangrikste en mees algemeen gebruikte swamdoder wat teen Penicillium groen- en blouskimmel toegedien word (Figuur 1 en 2, onderskeidelik). ʼn Eïenskap wat dit meer gunstig as ander Penicillium swamdoders maak, is sy vermoë om sporulasie te onderdruk (Figuur 3). Die voortdurende gebruik van IMZ maak die ontwikkeling van IMZ weerstandbiedenheid in Penicillium populasies onafwendbaar. Die tempo waarteen weerstandbiedenheid ontwikkel kan egter verminder word deur verskillende swamdoder-aktiewes teen Penicillium, toe te dien, en belangrikste, die inokulumlading in die pakhuis-omgewing moet deur effektiewe sanitasie-praktyke verminder word. Dit voorkom die seleksie en opbou van weerstandbiedende Penicillium isolate. Die effektiwiteit van ʼn spesifieke sanitasie protokol kan in die korttermyn bepaal word deur die vang en tel van spore (konidia) in die water, lug en op oppervlaktes vóór en ná ʼn spesifieke sanitasie-aksie. Indien die tweede spoortelling minder is as die eerste, was die sanitasie-aksie effektief. Die frekwensie waarteen swamdoder weerstandbiedenheid voorkom moet oor die medium- tot langtermyn gedurende die pakseisoen bepaal word. Dit moet dus elke seisoen herhaal word ten einde ʼn goeie aanduiding van die pakhuis se spesifieke weerstandsprofiel te verkry. Huidige diagnostiese protokolle laat slegs vir die waarneem van weerstandbiedenheid toe, en nie vir kwantifisering van weerstandbiedenheid nie. Met ander woorde, ons kan slegs waarneem of weerstandbiedende individue op ʼn spesifieke tyd teenwoordig is of nie, maar ons weet nie hoeveel weerstandbiedende individue teenwoordig is nie. Ten einde die vlak van weerstandbiedende individue te kwantifiseer, sal ʼn intensiewe opname vereis word, wat ʼn groot aantal metings op ʼn meer gereelde basis sal behels.dit sal te duur wees om as ʼn roetinetoets uitgevoer te word. CRI gaan voort om ʼn navorsingsprojek by die Universiteit van Stellenbosch te befonds, wat ten doel het om nuwe tegnologieë te ontwikkel wat die vinnige en akkurate vasstelling van die IMZ weerstandbiedenheidsfrekwensie in pakhuise moontlik sal maak. In die tussentyd moedig ons pakhuise aan om hul IMZ weerstandbiedenheidstatus (teenwoordig of afwesig) ten minste 3 keer ʼn seisoen te bepaal. Metode. Die CRI Diagnostiese Sentrum (DC) het ʼn IMZ weerstandbiedenheidswaarneming bepalingsmetode beskikbaar. Dit kan vanaf spoorvangers (Figuur 4) óf dempers (Figuur 5) gedoen word. ʼn Spoorvanger bestaan uit ʼn Petri bakkie bevattende ʼn filtreerpapierskyfie. Die filtreerpapierskyfie word met ʼn kleefmiddel behandel ten einde te verseker dat gevangde spore op die filtreerpapier bly. Hierdie metode is ideaal vir die vang van spore in die lug. Dempers kan gebruik word om spore vanaf oppervlaktes of verrotte (sporulerende) vrugte op tel. Spoorvangers kan vanaf die DC bestel word. Dempers kan by die meeste apteke of by jou chemiese verteenwoordiger gekoop word. Instruksies vir die gebruik van spoorvangers Voltooi die etiket op al 4 Petri bakkies. Vul asseblief jou kontak-inligting in sodat jou pakhuisresultate na jou ge-e-pos kan word. JOU HEFFING WERK VIR JOU PRODUSENTE SE HEFFINGS WORD AANGEWEND OM DIE AKTIWITEITE VAN DIE CRI TE BEFONDS 1

2 CRI Snykant NAVORSINGSNUUS VAN CITRUS RESEARCH INTERNATIONAL Julie 2014 Nr 183 Plaas een spoorvanger op elk van die volgende areas in the pakhuis: 1. Ontgroeningskamer 2. Kantel-area 3. Wakstoediener 4. Verpakkingsarea Die spoorvanger moet 1 m bokant die grond geplaas word, weg van moontlike vloeistofsproei, ens. Dit is baie belangrik dat die Petri bakkie nie nat word nie. o ʼn Goeie plek is om die spoorvanger op ʼn raamwerk bokant die aangewese area te plaas. Maak die Petri bakkie oop en plaas die deksel onder die bakkie. JOU HEFFING WERK VIR JOU PRODUSENTE SE HEFFINGS WORD AANGEWEND OM DIE AKTIWITEITE VAN DIE CRI TE BEFONDS 2

3 CRI Snykant NAVORSINGSNUUS VAN CITRUS RESEARCH INTERNATIONAL Julie 2014 Nr 183 Los die spoorvanger oop vir 7 dae. Maak die spoorvanger toe en skryf die datum en tyd neer wat dit toegemaak is. Seël die spoorvangerbakkie met kleefband of Parafilm. Stuur die spoorvangers binne verskafde houers na die CRI se DC vir ontleding. Moet nie oop spoorvangers (sonder deksels) vir bepaling stuur nie, dit sal nie ʼn akkurate resultaat lewer nie en sal nie deur die DC aanvaar word nie. Aanbeveling vir gebruik van ʼn demper o Sporulerende vrugte Verrotte raklewe vrugte (teruggehoude vrugte). Afgekeurde vrugte weens verval. WAARSKUWING! Moet nie die demper binne-in die vrot vrug steek nie. Die doel is om slegs die groenof blouskimmelspore op te tel en nie spore van ander skimmels nie. Raak liggies aan die sporulerende oppervlak ten einde slegs die groen of blou spore op te tel (Figure 7 9). Plaas die demper dadelik terug in sy kapsule (Figure 10 & 11) o Moenie oop dempers vir bepaling stuur nie, dit sal nie ʼn akkurate resultaat lewer nie, en sal nie deur die DC aanvaar word nie. Dempers moet verkieslik vir beskerming in ʼn borrel koevert gestuur word. JOU HEFFING WERK VIR JOU PRODUSENTE SE HEFFINGS WORD AANGEWEND OM DIE AKTIWITEITE VAN DIE CRI TE BEFONDS 3

4 CRI Snykant NAVORSINGSNUUS VAN CITRUS RESEARCH INTERNATIONAL Julie 2014 Nr 183 By die DC sal spore vanaf die spoorvangers of dempers na water oorgedra word. ʼn Verdunningsreeks sal op ADA (groeimedium) uitgeplaat word by die helfte van die ADA sal IMZ gevoeg word. Indien Penicillium spore op die aangepaste medium groei, sal dit as weerstandbiedend beskou word. Weens die uitplaatproses en die tyd wat dit neem vir kulture om te groei, sal dit twee tot vier weke neem vanaf die inname van monsters by die DC, tot die uitstuur van die verslag. Spoorvangers kan vanaf die diagnostikus, Elaine Basson, bestel word, óf telefonies ( ) óf deur e-pos Elke spoorvanger sal ook ʼn etiket hê, wat deur die pakhuis voltooi moet word. Die koste van een spoorvanger is R 7.00 (BTW en versending uitgesluit). ʼn Aansoek vir analise vorm sal by elke besending van spoorvangers ingesluit word. Die vorm moet deur die pakhuis voltooi word en saam met die spoorvangers teruggestuur word. Indien dempers gestuur word, kan die vorm vanaf die CRI se webtuiste afgelaai word ( ostic) of kontak die diagnostikus. Die koste vir analise van een monster is R185. Indien meer as 4 monsters vanaf ʼn pakhuis gestuur word, sal die koste 4 x R185 ( = R740) wees, plus R70 vir elke addisionele monster. Alle pryse sluit BTW uit. JOU HEFFING WERK VIR JOU PRODUSENTE SE HEFFINGS WORD AANGEWEND OM DIE AKTIWITEITE VAN DIE CRI TE BEFONDS 4

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6 CRI Cutting Edge RESEARCH NEWS FROM CITRUS RESEARCH INTERNATIONAL July 2014 No 183 Assessing the incidence of imazalil resistance in a citrus packhouse environment by Arno Erasmus a, Elaine Basson a, Mareli Kellerman ab, Catherine Savage a, Keith Lesar a, Cheryl Lennox b and Paul Fourie ab a Citrus Research International, Nelspruit b Department Plant Pathology, University Stellenbosch Background. Imazalil (IMZ) is currently our most important and frequently used fungicide applied against Penicillium green and blue mould (Figure 1 and 2, respectively). An attribute that makes it more favourable than other Penicillium fungicides, is its ability to inhibit sporulation (Figure 3). The prolonged use of IMZ makes development of IMZ resistance in Penicillium populations inevitable. However, the rate of resistance development can be reduced by applying a variety of different fungicides active against Penicillium and most importantly the inoculum load in the packhouse environment must be reduced through effective sanitation practices. This prevents the proliferation of resistant Penicillium isolates. In the short term the efficacy of a specific sanitation regime can be assessed by capturing and counting spores (conidia) in the water, air and on surfaces before and after a specific sanitation action. If the second spore count is less than the first, the sanitation action has been effective. In the medium to long term the fungicide resistance frequency should be assessed during the packing season and this should be repeated every season in order to get a good indication of the packhouse s specific resistance profile. Current diagnostic protocols only allow for the detection of resistance, and not for quantification of that resistance. In other words, we can only detect whether resistant individuals are present at a certain time or not, but we don t know how many resistant individuals are present. To quantify the level of resistant individuals will require a very intensive assessment, which will entail a high number of assessments on a more frequent basis, which will be too costly to conduct as a routine test. CRI is continuing to fund a research project at Stellenbosch University that aims to develop new technologies that should enable the rapid and accurate determination of the IMZ resistance frequency in packhouses. In the meantime, we encourage packhouses to determine their IMZ resistance status (presence or absence) at least 3 times a season. Method. The CRI Diagnostic Centre (DC) has an IMZ-resistance detection assessment available. This can be done by using either spore traps (Figure 4) or swabs (Figure 5). A spore trap consists of a petri dish containing a filter paper disc. The filter paper disc is treated with an adhesive to ensure that the captured spores stay on the filter paper. This method is ideal for trapping spores in the air. Swabs can be used to pick up spores from surfaces or rotten (sporulating) fruit. Spore traps can be ordered from the DC. Swabs can be bought at most pharmacies or from your chemical representative. Instructions for using spore traps Complete the label on all 4 of the Petri dishes. Please fill in your contact details so that we may your packhouse s results to you. YOUR LEVY WORKING FOR YOU GROWER LEVIES ARE USED TO FUND THE ACTIVITIES OF CRI 1

7 CRI Cutting Edge RESEARCH NEWS FROM CITRUS RESEARCH INTERNATIONAL July 2014 No 183 Place one spore trap at each of the following locations in the packhouse: 1. Degreening room 2. Tipping area 3. Wax applicator 4. Packing area The spore trap should be placed 1 m above the floor, away from possible liquid spills etc. It is very important that the Petri dish does not get wet. o A good place is to place the trap on a lattice above the designated area. Open the Petri dish and place the lid underneath the dish. YOUR LEVY WORKING FOR YOU GROWER LEVIES ARE USED TO FUND THE ACTIVITIES OF CRI 2

8 CRI Cutting Edge RESEARCH NEWS FROM CITRUS RESEARCH INTERNATIONAL July 2014 No 183 Leave the trap open for 7 days. Close the trap and write the date and time it was closed. Seal the trap dish with Sellotape or Parafilm. Send the traps inside the provided containers to the CRI DC for assessment Do not send open spore traps (without lids) for assessment, this will not give an accurate result and will not be accepted by the DC. Recommendation for using a swab o Sporulating fruit Rotten shelf life fruit (hold back fruit) Rejected fruit due to decay CAUTION! Do not stick the swab inside the rotten fruit. The aim is to only pick up the green mould spores and not from other moulds Lightly touch the sporulating surface to pick up only the green or blue spores (Figures 7 9) Swiftly place the swab back into its holding capsule (Figures 10 & 11) o Do not send open swabs for assessment, this will not give an accurate result and will not be accepted by the DC. Swabs should preferably be sent within a bubble envelope for protection. YOUR LEVY WORKING FOR YOU GROWER LEVIES ARE USED TO FUND THE ACTIVITIES OF CRI 3

9 CRI Cutting Edge RESEARCH NEWS FROM CITRUS RESEARCH INTERNATIONAL July 2014 No 183 At the DC, spores will be transferred from the traps or swabs to water. A dilution series will be plated out on PDA (growth medium) half of the PDA will be amended with IMZ. If Penicillium spores grow on the amended media it will be regarded as resistant. Due to the process of plating out and growing of cultures it will take two to four weeks from intake of samples at the DC to the sending of the report. Spore traps can be ordered from the diagnostician, Elaine Basson, either telephonically ( ) or by (elaineb@cri.co.za). Each spore trap will also have a label, which should be completed by the packhouse. The cost of one spore trap is R 7.00 (excl. VAT and postage). A request for diagnostic analysis form will be included with each consignment of spore traps. The form needs to be completed by the packhouse and sent back with the spore traps. If swabs are sent, please download the form from CRI s website: ( ostic) or contact the diagnostician. The cost of analyses for one sample is R185. If more than 4 samples are submitted from a packhouse, the cost will be 4 x R185 ( = R740) plus R70 for every additional sample. All prices exclude VAT. YOUR LEVY WORKING FOR YOU GROWER LEVIES ARE USED TO FUND THE ACTIVITIES OF CRI 4