Epidemiology and management of flower diseases of pyrethrum

Pyrethrum (Tanacatum cinerariifolium) is cultivated worldwide and in southern Australia for the extraction of insecticidal esters or pyrethrins contained within the achenes of flowers. Producing a significant proportion of the worlds botanical pyrethrins, Australian crops may suffer reduced yields from annual flower disease epidemics caused by pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Little is known regarding (i) the epidemiology of flower blights caused by B. cinerea and S. sclerotiorum in pyrethrum, (ii) the efficacy of current control methods, (iii) whether there is evidence of fungicide resistance in the fungal population, (iv) whether alternative fungicides can provide improved control over those currently used, (v) whether other fungi could be involved in annual flower disease epidemics and, (vi) the loss in flower yield and pyrethrin assay from flower diseases. A survey of the incidence of B. cinerea and S. sclerotiorum in flowers was undertaken in ten commercial pyrethrum crops in one year. Flowers from non-fungicide treated areas in commercial crops were periodically sampled throughout flowering prior to being surface sterilised and incubated under high humidity to encourage fungal growth. B. cinerea and S. sclerotiorum were prevalent, with both occurring in flowers from all 10 crops. The mean incidence of B. cinerea in flowers sampled between 10-11 December across all crops was 56%, significantly higher (P = 0.024) than S. sclerotiorum (29.4%), while between 16-18 December mean incidence of B. cinerea was 75.7%, again significantly higher (P = 0.026) than incidence of S. sclerotiorum (51.8%) at this time. The main means of managing flower blights of pyrethrum is currently through a fungicide program over flowering involving tebuconazole and carbendazim. The efficacy of the flowering fungicide program for controlling B. cinerea and S. sclerotiorum flower blights and promoting benefits in terms of increased yield was evaluated in nontreated and fungicide treated plots in 10, 10 and 17 commercial pyrethrum crops during the flowering period over three years, respectively. In each of two years, fungicide treatment resulted in a mean incidence of B. cinerea near to harvest which bordered on being significantly lower (0.05 < P < 0.1) than no treatment. In the third year, fungicide treatment significantly (P = 0.038) reduced mean fungal incidence from 75% to 53.3% in nontreated close to harvest. The incidence of Sclerotinia sclerotiorum in flowers from nontreated was significantly higher (P < 0.001) than from fungicide treated in year one (62.8% and 29.2% respectively) and again significantly higher (P = 0.019) in year two (26% and 8.1% respectively). Across all fields, fungicide applications in year one resulted in no significant yield improvement. During year two, mean pyrethrin yield (across all sites) was significantly increased from 90.4kg/ha in nontreated areas to 108.4 kg/ha in fungicide treated plots (P = 0.032) and during the final year fungicide treatment significantly increased dry flower yield by 216 kg/ha (P = 0.018) in comparison to nontreated. However, while fungal incidence was often reduced within individual fields or across all sites by fungicides, few measurable benefits in yield quantity or quality were measured. In vitro sensitivity testing of 49 and 96 isolates of B. cinerea and S. sclerotiorum respectively to fungicides commercially used for control was conducted. A proportion of B. cinerea isolates appeared sensitive to carbendazim at low concentrations i.e. 39% had EC50 less than 1.05 ˜í¬¿g a.i./ml. However a high proportion (61%) of B. cinerea isolates had EC50 values to carbendazim greater than 100 ˜í¬¿g a.i./ml. This provided evidence for potential resistance amongst the population of B. cinerea and may explain the inconsistency of yield responses from the fungicide program. By comparison, 96.9% of S. sclerotiorum isolates had EC50 values <2.4 ˜í¬¿g a.i./ml to carbendazim with a mean of 0.5 ˜í¬¿g a.i./ml. However, a small number (3.1%) of S. sclerotiorum isolates were highly insensitive to carbendazim with EC50 of >1000 ˜í¬¿g a.i./ml. All isolates of B. cinerea and S. sclerotiorum were sensitive to tebuconazole at low concentrations with mean EC50 of 0.64 and 0.18 ˜í¬¿g a.i./ml respectively. Alternative fungicides for flower disease control were evaluated in replicated field trials to determine suitability for inclusion into the flowering fungicide program as replacements for the potentially ineffective and now deregistered fungicide carbendazim. Boscalid and iprodione showed greater benefits in terms of yield from statistical analysis than other treatments, and significantly reduced fungal incidence of flowers equal to, or better than, commercial treatments. The mean incidence of B. cinerea from nontreated flowers sampled on 4 December of 30% was significantly (P = 0.007) higher than boscalid (8.5%) and iprodione (10%). Mean incidence of S. sclerotiorum of flowers from nontreated plots sampled on 4 December was 26.5%, and significantly (P <.001) higher than boscalid (7%) and iprodione (12%). In vitro fungicide sensitivity of 46 isolates of B. cinerea and S. sclerotiorum to iprodione indicated no evidence of reduced sensitivity with mean EC50 (and maximum) values of 1.62 (8.48 ˜í¬¿g a.i./ml) and 0.19 (0.61 ˜í¬¿g a.i./ml) respectively. Sclerotinia minor, a previously undocumented pathogen of pyrethrum flowers, was consistently isolated from diseased, surface-sterilised pyrethrum flowers over multiple years. Fungal identity was confirmed with morphological, genetic and phylogenetic evaluation. Occurrence in-field of mature, sporulating, apothecia of S. minor were documented; while sclerotia of 8 of 10 isolates conditioned in the laboratory successfully underwent carpogenic germination in vitro demonstrating the ability of endemic isolates of S. minor to produce air borne inoculum necessary to achieve flower infection in pyrethrum and other crops. The relative sensitivity of isolates of S. minor to the fungicides carbendazim, tebuconazole and iprodione was evaluated. Mean (and maximum) EC50 values of S. minor were 1.92 (2.62 ˜í¬¿g a.i/ml) for carbendazim, 0.1 (0.13 ˜í¬¿g a.i./ml) for tebuconazole and 0.3 (1.23 ˜í¬¿g a.i/ml) for iprodione. The effect of B. cinerea and S. sclerotiorum flower inoculation on measured yield attributes and pyrethrum flower development were evaluated with glasshouse studies. Inoculation of immature flowers with Botrytis cinerea resulted in highly significant reductions in dry weight (P <.001) and pyrethrin yield (P <.001) of mature flowers compared to non inoculated. Inoculation with S. sclerotiorum in two of four varieties resulted in significantly higher flower development stages after 21 days (P = 0.021; P <.001) and significantly faster flower senescence (P = 0.001; P = 0.008) in comparison to non inoculated. Inoculation of flowers with ascospores of S. minor led to symptoms of flower disease indistinguishable from those of S. sclerotiorum flower blight, significantly lower fresh flower weights in replicated experiments (P = <.001; 0.032) and significantly higher developmental stage of flowers (P = <.001) after only two weeks in comparison to non inoculated. These studies indicated that flower infection with B. cinerea and S. minor could significantly reduce flower yield and pyrethrin yields. Furthermore, significantly faster flower development; senescence and desiccation may result from flower infection with S. sclerotiorum and S. minor. The completion of Koch's postulates additionally confirmed pathogenicity toward pyrethrum flowers and demonstrated pure cultures of S. minor could be reisolated from flowers inoculated on living host plants. The study has provided new knowledge in the epidemiology of known flower blights of pyrethrum caused by B. cinerea and S. sclerotiorum. Furthermore the study has described for the first time S. minor as a pathogen of pyrethrum flowers and documented a rare example of carpogenic germination of sclerotia being involved in the epidemiology of a disease caused by S. minor. The study has also provided insights into the effect of flower blights on pyrethrum yield and into improving the management of flower diseases by fungicides.