Biological activity of Pyraclostrobin against Coniella granati causing pomegranate crown rot

Pomegranate crown rot caused by Coniellagranati is one of the most severe diseases of pomegranate. To date, no fungicides have been registered for controlling this disease in China. Pyraclostrobin, belonging to strobilurin fungicides, has a broad spectrum of activity against many phytopathogens. In this study, based on the mycelial growth and conidial germination inhibition methods, we investigated the biological activity of pyraclostrobin against C. granati at the presence of 50 μg/mL SHAM using 80 isolates collected from different orchards in China during 2012-2018. The EC50 (50% effective concentration) values ranged from 0.040-0.613 μg/mL for mycelial growth and 0.013-0.110 μg/mL for conidium germination, respectively. Treated with pyraclostrobin, the hyphae morphology changed and conidial production of C. granati decreased significantly. The result of transmission electron microscope showed that treatment of pyraclostrobin could make the cell wall thinner, and lead to ruptured cell membrane and formation of intracellular organelle autophagosomes. The pyraclostrobin showed good protective and curative activities against C. granati on detached pomegranate fruits. In field trials, pyraclostrobin showed excellent control efficacy against this disease in which the treatment of 25% pyraclostrobin EC 1000× provided 92.25% and 92.58% control efficacy in 2019 and 2020, respectively, significantly higher than that of other treatments. Therefore, pyraclostrobin could be a candidate fungicide for the control of pomegranate crown rot.

Antifungal activity of hydroxytyrosol enriched extracts from olive mill waste against Verticillium dahliae, the cause of Verticillium wilt of olive

Verticillium wilt (caused by Verticillium dahliae Kleb.) is an important disease affecting olive (Olea europaea L.) production. Effective control of this disease relies on integrated management strategies. In vitro antifungal activity of two hydroxytyrosol (HTyr) enriched extracts (HTE1 and HTE2) obtained from olive mill waste products (OMWP) was assessed against V. dahliae. Inhibitory effects of pure HTyr as a standard, and HTE1 and HTE2 at different concentrations, were evaluated on mycelium growth and conidium germination of V. dahliae. Chemical characterization of HTE1 and HTE2 allowed identification and quantification of HTyr as the main constituent in both extracts along with other low molecular weight phenols. HTE1 showed a higher inhibitory activity in both growth and conidium germination of V. dahliae. At the tested concentrations, low antifungal effects of HTyr were observed. After 3 d, 1 mg mL-1 of HTE1 gave greater inhibition of mycelium growth than HTE2 or HTyr. After 24 h, HTE1 gave 55% inhibition of conidium germination, and HTyr and HTE2 both gave 37% inhibition. This study has demonstrated that phenolic compounds derived from OMWP have antifungal activity against V. dahliae, indicating the potential of these compounds for eco-friendly control of Verticillium wilt.

Temperature and incubation period affect Septoria pistaciarum conidium germination: disease forecasting and validation

Septoria leaf spot is an important disease of pistachio trees in Greece. This study aimed to determine effects of temperature and the incubation period on germination of conidia of Septoria pistaciarum, and to evaluate a generic model to forecast pistachio leaf spot under the field conditions of Aegina Island, Greece. The optimum temperature for conidium germination was 23°C, and germination was inhibited at 35 and 4°C. At constant temperature of 23°C, conidia commenced germination after 9 h. The predictive model indexed disease risk close to 100 at 10 May at two locations (Rachi Moschona and Vigla) in 2017, and first leaf spot symptoms were observed on 17 May. Moderate to high disease severity (>25% leaves infected) were observed in unsprayed trees at the end of May. In 2018, the model indexed risk close to 100 on 9 May at Rachi Moschona, and first symptoms were observed on 18 May. Moderate to high disease severity (>25% leaves infected) were observed in unsprayed trees on 25th of May. This study has shown that the forecasting model can be used in Aegina Island, Greece, to predict the severity of Septoria leaf spot of pistachio.

Transcription Factor CfSte12 of Colletotrichum fructicola Is a Key Regulator of Early Apple Glomerella Leaf Spot Pathogenesis

ABSTRACT Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a rapidly emerging disease leading to defoliation, fruit spot, and storage fruit rot on apple in China. Little is known about the mechanisms of GLS pathogenesis. Early transcriptome analysis revealed that expression of the zinc finger transcription factor Ste12 gene in C. fructicola (CfSte12) was upregulated in appressoria and leaf infection. To investigate functions of CfSte12 during pathogenesis, we constructed gene deletion mutants (ΔCfSte12) by homologous recombination. Phenotypic analysis revealed that CfSte12 was involved in pathogenesis of nonwounded apple fruit and leaf, as well as wounded apple fruit. Subsequent histological studies revealed that loss of pathogenicity by ΔCfSte12 on apple leaf was expressed as defects of conidium germination, appressorium development, and appressorium-mediated penetration. Further RNA sequencing-based transcriptome comparison revealed that CfSte12 modulates the expression of genes related to appressorium function (e.g., genes for the tetraspanin PLS1, Gas1-like proteins, cutinases, and melanin biosynthesis) and candidate effectors likely involved in plant interaction. In sum, our results demonstrated that CfSte12 is a key regulator of early apple GLS pathogenesis in C. fructicola. In addition, CfSte12 is also needed for sexual development of perithecia and ascospores. IMPORTANCE Glomerella leaf spot (GLS) is an emerging fungal disease of apple that causes huge economic losses in Asia, North America, and South America. The damage inflicted by GLS manifests in rapid necrosis of leaves, severe defoliation, and necrotic spot on the fruit surface. However, few studies have addressed mechanisms of GLS pathogenesis. In this study, we identified and characterized a key pathogenicity-related transcription factor, CfSte12, of Colletotrichum fructicola that contributes to GLS pathogenesis. We provide evidence that the CfSte12 protein regulates many important pathogenic processes of GLS, including conidium germination, appressorium formation, appressorium-mediated penetration, and colonization. CfSte12 also impacts development of structures needed for sexual reproduction which are vital for the GLS disease cycle. These results reveal a key pathogenicity-related transcription factor, CfSte12, in C. fructicola that causes GLS.

Effects of Temperature and Moisture on Conidia Germination, Infection and Acervulus Formation of Apple Marssonina Leaf Blotch Pathogen (Diplocarpon mali) in China

Apple marssonina leaf blotch (Diplocarpon mali) is a severe disease of apple that mainly causes premature leaf defoliation in many apple growing areas in the word. Its epidemic development is closely related to temperature and rainfall. The effects of temperature and moisture on conidium germination, infection on leaves, and acervulus production were investigated under controlled environments. Temperature required for conidium germination and infection ranged from 5 oC to 30 oC with the optimum around 23 oC, and temperature required for acervulus formation was slightly wider with the optimum at 24.6 oC. Wetness was needed for conidia to germinate and infect; only few conidia germinated at RH=100%. However, lesions can produce acervuli in dry conditions. The minimum duration of leaf wetness required for conidia to complete the entire infection process were 14, 8, 4, and 6 hours at 10, 15, 20, and 25°C, respectively. A model describing the effect of temperature and leaf wetness duration was built. The model estimated that conidial infection has the optimum temperature at 22.6°C and the minimum wetness duration required of 4.8 hours. The model can be used to forecast the conidial infection of D. mali to assist in disease management in commercial apple production.

The Endophytic Fungus Albifimbria verrucaria from Wild Grape as an Antagonist of Botrytis cinerea and Other Grape Pathogens

Gray mold, caused by Botrytis cinerea, is one of the most prevalent fungal diseases in table and wine grapes, affecting grape quality and yields. In this study, we isolated several endophytic fungi, including Alternaria alternata, Bipolaris cynodontis, Phoma sp., and Albifimbria verrucaria, from leaves of Amur grape (Vitis amurensis) cultivar Shuangyou and investigated their biocontrol activity against B. cinerea. In vitro dual assay showed that A. verrucaria isolate SYE-1 inhibited growth of B. cinerea. The isolate also had a wide range of biocontrol activity against Lasiodiplodia theobromae and Elsinoë ampelina. Mycelial growth and conidium germination of B. cinerea were significantly inhibited by metabolites of A. verrucaria in agar plates and culture extracts of A. verrucaria from liquid culture. The isolate produced a total chitinase activity of 0.4 U/ml after incubation for 10 days in Czapek’s liquid medium. In addition, application of culture extracts of A. verrucaria prior to B. cinerea inoculation significantly reduced disease severity on grape leaves of the susceptible cultivar Red Globe. Taken together, our results indicate that A. verrucaria has potential as a biocontrol agent to control grape gray mold.

Calcium Signaling Is Suppressed in Magnaporthe oryzae Conidia by Bacillus cereus HS24

Rice yield is greatly reduced owing to rice blast, a polycyclic fungal disease caused by the ascomycete Magnaporthe oryzae. Previously, Bacillus cereus HS24, isolated from a rice farm, showed a strong antimicrobial effect toward M. oryzae. To better exploit it as a biocontrol agent, HS24 was studied for the mechanism that it uses to suppress rice blast. Conidium germination in M. oryzae was significantly inhibited by HS24, whereby inhibition reached 97.8% at the concentration of 107 CFU/ml. The transcription levels of Ca2+/calmodulin-dependent protein kinase II, PMC1, and CCH1, key genes involved in the M. oryzae Ca2+ signaling pathway, were significantly decreased in HS24-treated conidia at high concentration. The treatment of M. oryzae with the corresponding Ca2+ signaling pathway inhibitors KN-93, verapamil, and cyclopiazonic acid significantly reduced conidium germination. This inhibitory effect was found to be concentration dependent, similar to the HS24 treatment. We also found that HS24 was able to decrease the intracellular free Ca2+ concentration in M. oryzae conidia significantly. The addition of exogenous Ca2+ did not diminish the inhibitory effect of HS24 on the reduction of intracellular free Ca2+ concentration and the level of conidium germination. In conclusion, B. cereus HS24 at high concentration prevents extracellular Ca2+ from entering the conidia in M. oryzae, causes a significant reduction of intracellular free Ca2+ concentration, and results in the inhibition of conidium germination.

A Threshold-Based Decision-Support System for Fungicide Applications Provides Cost-Effective Control of Citrus Postbloom Fruit Drop

Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum sensu lato and C. gloeosporioides sensu lato, is an important disease in the humid tropics of the American continent. PFD mainly affects flowers, on which typical symptoms are characterized by orange-brown lesions with presence of acervuli. The disease has a sporadic occurrence, but preventative fungicide sprays are applied every season. The objective of this study was to evaluate the effectiveness of a fungicide spray strategy for PFD based on a predictive model of C. acutatum conidium germination linked to weather conditions. Fungicide sprays were performed when the model predicted pre-established thresholds of 10, 15, 20, and 25% of germinated spores (T10, T15, T20, and T25, respectively). Five experiments were conducted in two different seasons in the state of São Paulo, Brazil. PFD control efficacy of the threshold-based treatments was compared with a nontreated control and to a calendar-based spray system. Additionally, an economic analysis was performed to assess the gross income revenues of the fungicide spraying strategies. Disease control in plots treated at T10, T15, and T20 was as effective as the calendar-based strategy. The number of fungicide applications was reduced by 33 to 71% when sprays were applied at T15 and T20, and gross income increased or was comparable to that of the other treatments. Therefore, using a conidium germination model with a threshold of 15 or 20% is recommended as a spraying strategy for PFD management in Brazil.