Mancozeb rainfastness and residue thresholds for control of Venturia inaequalis

Rossouw, Cornelis Johannes (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH SUMMARY: An important apple disease world-wide is Venturia inaequalis that causes scab-like lesions on fruit and defoliation of trees. In South Africa, the disease is mainly managed through fungicide sprays that mostly consist of the contact fungicide mancozeb. The volume and intensity of rainfall can influence the efficacy and persistence of mancozeb depositions. Mancozeb spray deposition can be assessed through expensive quantification of Manganese-ion residues, or a cost effective fluorescent pigment of which the coverage is accessed using photomacrography image analyses. The biological efficacy of quantitative mancozeb depositions can be determined through the development of benchmark models. For V. inaequalis, this is challenging since in vitro production of inoculum is difficult, and leaf lesions are slow to develop and not amendable to quantification through image analyses. The study showed that a yellow fluorescent pigment was a suitable tracer for five different mancozeb formulations (Dithane M-45 800 WP NT, Mancozeb 800 WP, Pennfluid, Ventum 800 WP, Vondozeb) since a good Pearson’s correlation (r = 0.779) existed between fluorescent particle coverage (FPC%) and mancozeb residues (Mn-ion (mg/kgDW)).The particle size ranges of Dithane M-45 800 WP NT and Ventum 800 WP were significantly smaller than those off the other formulations, but this did not result in differences in Mn-ion residues realized on apple leaves for WP formulations. The Pennfluid SC formulation deposited markedly less mancozeb than the WP formulations, due to a lower active ingredient label rate used. Rainfastness was evaluated for the Dithane M-45 800 WP NT and Ventum 800 WP formulations, and Ventum 800 WP combined with a sticker-spreader adjuvant Nu-Film P. Simulated rain applied to apple seedlings at a constant rainfall intensity of 5 mm/h at five different rainfall volumes (0, 1, 5, 10 and 15 mm) resulted in no significant differences between the three treatments, based on FPC% and Mn-ion concentrations. Although a good correlation (r = 0.726 to 0.783) existed between FPC% and Mn-ions, the response of FPC% and Mn-ion differed somewhat as was evident from the slopes of exponential regression models, showing slower initial loss in FPC% than for Mn-ions and a markedly larger predicted loss by the model’s asymptotic value (61.4% and 32.2%, respectively). A significant loss (11.95%) in mancozeb residue occurred after applying 1mm of rain, but no significant differences in losses (26.32 to 31.67%) occurred after applying 5 to 15mm of rain. Benchmark development for mancozeb deposition for the control of V. inaequalis involved apple seedling leaves being treated with a concentration range of mancozeb and fluorescent pigment (0, 0.15 ×, 0.3 ×, 0.45 ×, 0.6 × and 1.0 ×), followed by inoculation with conidia harvested from naturally infected orchard leaves. Venturia inaequalis control was assessed using a basic fuschin based staining technique and visual assessment. The staining technique was useful for quantifying infection within 6 days, but underestimated percentage control relative to the visual assessment of lesions after 3-4 weeks. Complete control was observed for all mancozeb concentrations based on visual lesion assessment. No function could thus be fitted to deposition quantity data (0.29 to 8.28 FPC% values) versus disease control (staining or visual assessment). The cellophane agar plate technique was optimized for the in vitro production of V. inaequalis conidium inoculum that can be used in future infection studies. After 1 week, optimum spore production (1.59× 106 conidia/ml) and viability (± 85%) were observed that were significantly higher than at weeks 2 to 4. The study provided valuable information for the assessment of mancozeb deposition, mancozeb rainfastness, and for benchmark model development with regards to the rapid quantification of lesions on leaves and the in vitro production of V. inaequalis inoculum. The yellow fluorescent pigment can now be used as an excellent cost effective tracer for mancozeb deposition of various formulations on apple seedling leaves, and will also be helpful in identifying trends on the effect of rain on the persistence of mancozeb formulations. This has increased research capacity towards evaluating the rainfastness of fungicides using simulated rain, and orchard trials for accessing the effect of spray volumes and machines through fluorescent pigment deposition. Although a benchmark model could not be developed, the development of a model in future will be more feasible since a rapid staining technique for quantification of V. inaequalis disease severity was identified that will just have to be optimized further. Secondly, a cellophane agar plate technique, and isolates with high spore production capacity for axenic conidial production will further facilitate model development.

AFRIKAANSE OPSOMMING: Venturia inaequalis is ‘n belangrike appelsiekte wêreldwyd, wat skurfagtige letsels op vrugte en die afval van blare veroorsaak. In Suid-Afrika word die siekte hoofsaaklik deur die spuit van fungisiedes bestuur, wat grootliks uit die kontakfungisied, mankozeb, bestaan. Die volume en intensiteit van reënval kan die doeltreffendheid en werkingsduur van mankozeb deponerings beïnvloed. Mankozeb spuitdeponerings kan deur duur kwantifisering van mangaan-ioon residue bepaal word, of deur ‘n koste-effektiewe fluoresserende pigment waarvan die bedekking bepaal word deur die gebruik van fotomakrografie beeld-analise. Die biologiese doeltreffendheid van kwantitatiewe mankozebdeponerings kan bepaal word deur die ontwikkeling van basisvlak modelle. Vir V. inaequalis is dit ‘n uitdaging aangesien in vitro produksie van inokulum moeilik is, en blaarletsels stadig ontwikkel, en nie geskik is vir kwantifisering deur beeld-analise nie. Die studie het getoon dat ‘n geel fluoresserende pigment ‘n geskikte aanwyser van vyf verskillende mankozeb formulasies was (Dithane M-45 800 WP NT, Mancozeb 800 WP, Pennfluid, Ventum 800 WP, Vondozeb), aangesien ‘n goeie Pearson korrelasie (r = 0.779) tussen fluoresserende partikel bedekking (FPC%) en mankozeb residue (Mn-ioon (mg/kgDW)) bestaan het. Die partikel grootte reeks van Dithane M-45 800 WP NT en Ventum 800 WP was betekenisvol kleiner as dié van die ander formulasies, maar dit het nie tot verskille in Mn-ioon residue, soos verkry op appelblare vir WP formulasies, gelei nie. Die Pennfluid SC formulasie het merkbaar minder mankozeb as die WP formulasies gedeponeer, weens ‘n laer aktiewe bestanddeel etiket toedienings dosis wat gebruik is. Reënvastheid is vir die Dithane M-45 800 WP NT en Ventum 800 WP formulasies, en Ventum 800 WP, gekombineer met ‘n kleefmiddel-verspreider adjuvant, Nu-Film P, geëvalueer. Gesimuleerde reën wat tot appelsaailinge teen ‘n konstante reënval-intensiteit van 5 mm/h by vyf verskillende reënvalvolumes (0, 1, 5, 10 en 15 mm) toegedien is, het tot geen betekenisvolle verskille tussen die drie behandelings gelei nie, gebaseer op FPC% en Mn-ioon konsentrasies. Hoewel ‘n goeie korrelasie (r = 0.726 tot 0.783) tussen FPC% en Mn-ione bestaan het, het die reaksie van FPC% en Mn-ioon ietwat verskil, soos wat duidelik was vanuit die kurwes van eksponensiële regressie modelle, wat stadiger aanvanklike verlies in FPC% as vir Mn-ione getoon het, en ‘n merkbaar groter voorspelbare verlies deur die model se asimptotiese waarde (onderskeidelik 61.4% en 32.2%). ‘n Betekenisvolle verlies (11.95%) in mankozeb residue het ná die toedien van 1mm reën voorgekom, maar geen betekenisvolle verskille in verliese (26.32 tot 31.67%) het voorgekom ná die toedien van 5 tot 15mm reën nie. Basisvlak ontwikkeling vir mankozeb deponering vir die beheer van V. inaequalis het behels dat appelsaailingblare met ‘n konsentrasiereeks van mankozeb en fluoresserende pigment (0, 0.15 ×, 0.3 ×, 0.45 ×, 0.6 × and 1.0 ×) behandel is, gevolg deur inokulasie met konidia wat vanaf natuurlik geïnfekteerde boordblare geoes is. Venturia inaequalis beheer is bepaal deur die gebruik van ‘n basiese ‘fuschin’-gebaseerde kleurtegniek en visuele bepaling. Die kleurtegniek was bruikbaar vir die kwantifisering van infeksie binne 6 dae, maar het die persentasie beheer relatief tot die visuele bepaling van letsels ná 3-4 weke onderskat. Volledige beheer is vir alle mankozeb konsentrasies waargeneem, gebaseer op visuele letselbepaling. Geen funksie kon dus gepas word op deponering kwantiteit data (0.29 tot 8.28 FPC% waardes) teenoor siektebeheer (kleuring of visuele bepaling) nie. Die ‘cellophane’ agarplaattegniek is geoptimaliseer vir die in vitro produksie van V. inaequalis konidium-inokulum wat in toekomstige infeksie-studies gebruik kan word. Ná 1 week, is optimum spoorproduksie (1.59× 106 konidia/ml) en lewensvatbaarheid (± 85%) waargeneem, wat betekenisvol hoër was as by weke 2 tot 4. Die studie het waardevolle inligting verskaf vir die bepaling van mankozeb deponering, mankozeb reënvastheid, en vir basisvlak model-ontwikkeling met betrekking tot die vinnige kwantifisering van letsels op blare en die in vitro produksie van V. inaequalis inokulum. Die geel fluoresserende pigment kan nou as ‘n uitstekende koste-effektiewe aanwyser vir mankozeb, vir verskeie formulasies, op appelsaailingblare gebruik word, en sal ook handig wees in die identifisering van tendense van die effek van reën op die volhoubaarheid van mankozeb formulasies. Dit het navorsingskapasiteit verhoog in terme van die evaluering van reënvastheid van fungisiedes deur die gebruik van gesimuleerde reën, en boordproewe vir die bepaling van die effek van spuitvolumes en masjiene deur fluoresserende pigmentdeponering. Hoewel ‘n basisvlak model nie ontwikkel kon word nie, sal die ontwikkeling van ‘n model in die toekoms meer haalbaar wees, aangesien ‘n vinnige kleurtegniek vir die kwantifisering van die hoeveelheid V. inaequalis siekte, geïdentifiseer is, wat net verder geoptimaliseer moet word. Tweedens sal ‘n ‘cellophane’ agar plaattegniek, en isolate met hoë spoorproduksie kapasiteit vir in vitro konidia produksie, verdere model ontwikkeling aanhelp.

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