Deena Errampalli

Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.

Prunus domestica Pathogenesis-Related Protein-5 Activates the Defense Response Pathway and Enhances the Resistance to Fungal Infection

Pathogenesis-related protein-5 (PR-5) has been implicated in plant disease resistance and its antifungal activity has been demonstrated in some fruit species. However, their roles, especially their interactions with the other defense responses in plant cells, are still not fully understood. In this study, we have cloned and characterized a new PR-5 cDNA named PdPR5-1 from the European plum (Prunus domestica). Expression of PdPR5-1 was studied in different cultivars varying in resistance to the brown rot disease caused by the necrotrophic fungus Monilinia fructicola. In addition transgenic Arabidopsis, ectopically expressing PdPR5-1 was used to study its role in other plant defense responses after fungal infection. We show that the resistant cultivars exhibited much higher levels of transcripts than the susceptible cultivars during fruit ripening. However, significant rise in the transcript levels after infection with M. fructicola was observed in the susceptible cultivars too. Transgenic Arabidopsis plants exhibited more resistance to Alternaria brassicicola. Further, there was a significant increase in the transcripts of genes involved in the phenylpropanoid biosynthesis pathway such as phenylalanine ammonia-lyase (PAL) and phytoalexin (camalexin) pathway leading to an increase in camalexin content after fungal infection. Our results show that PdPR5-1 gene, in addition to its anti-fungal properties, has a possible role in activating other defense pathways, including phytoalexin production.

Control of blue mold caused by Penicillium expansum on apples 'Empire' with fludioxonil and cyprodinil

Penicillium expansum, one of the important causal agents of blue mold of apple in storages in Ontario, has developed resistance to thiabendazole, a postharvest fungicide, in some storages. In an attempt to develop alternate fungicides to thiabendazole, the protective effect of three fungicides, fludioxonil, cyprodinil, and a mixture of fludioxonil and cyprodinil, were evaluated for their efficacy against blue mold caused by thiabendazole-sensitive and -resistant isolates of P. expansum. The three fungicides were equally effective against thiabendazole-sensitive and -resistant isolates of P. expansum. Fenhexamid had no activity against P. expansum. No cross-resistance was observed between thiabendazole and fludioxonil or cyprodinil. Lower concentrations of fludioxonil (45 µg·mL–1), cyprodinil (50 µg·mL–1), and fludioxonil + cyprodinil (50 + 75 µg·mL–1) gave >97.0% control for up to 1 month, but higher concentrations of fludioxonil (100 µg·mL–1) and fludioxonil + cyprodinil (150 + 225 µg·mL–1) were required to control blue mold for 62 days at 4 °C. When the mixture was tested, antagonistic effect was observed at lower concentrations; however, higher concentrations completely controlled the disease. Fludioxonil and cyprodinil have modes of action different from each other and from that of the thiabendazole; they have the potential to be effective components in the resistance management for postharvest control of apple blue mold.

Expression analysis of a plum pathogenesis related 10 (PR10) protein during brown rot infection

Plant PR10 is one of the pathogenesis related proteins, induced upon exposure to different stress conditions including fungal infection. PR10 proteins have been implicated in fungal disease resistance in some species; however its transcriptional regulation is not well understood. In the present work we cloned a PR10 gene from European plums (Prunus domestica L.) and monitored the quantitative changes in its transcript levels as a result of fungal infection in two varieties. We also studied the possible involvement of the membrane degrading enzyme phospholipase D-alpha (PLDα). In the susceptible variety, ‘Veeblue’, infection with the brown rot fungus Monilinia fructicola induced PLDα and PR10 expression, while in the resistant variety, ‘Violette’, a constitutive expression of PLDα and PR10 transcripts levels were observed. Resistance to M. fructicola also coincides with a sharp decrease in the expression of ABI1, a protein phosphatase and elevated hydrogen peroxide content after infection. Further, inhibition of PLDα by hexanal treatment, up-regulated ABI1 and decreased PR10 expression, suggesting a possible relationship between the two. We further confirm these results in Arabidopsis abi1 mutant that shows a higher level of PR10 transcripts.

A PCR-based method for detection of potato pathogen, Helminthosporium solani, in silver scurf infected tuber tissue and soils.

Silver scurf caused by Helminthosporium solani causes significant economic losses in table stock, seed and processing potatoes. Specific polymerase chain reaction (PCR) primers, Hs1F1/Hs2R1, from H. solani were used for the amplification of a 447-bp product from 20 tissue samples and 54 single spore H. solani isolates, from eastern Canada (27 isolates), western Canada (13 isolates) and North Dakota in USA (14 isolates), but not from other potato fungal pathogens. In addition to PCR analysis, all 54 isolates were studied using conventional detection methods, visual disease symptoms and/or colony morphology and microscopic examination of the morphology of conidiophores and conidia. The PCR assay successfully detected H. solani and the PCR results correlated well with assessments based on conventional techniques. The detection of H. solani by PCR (1 day) is rapid and offers an alternative to the time consuming conventional diagnostic techniques (4-5 weeks). Nested PCR assay was necessary for the detection of H. solani in soils and thus can provide a sensitive technique to study the epidemiology of silver scurf in soils.

Penicillium expansum (Blue Mold)

Abstract Blue mold disease, caused by Penicillium expansum (Link), is the most economically important postharvest disease of fruit and vegetables in storage. In addition to causing food spoilage, some strains of the fungus produce the mycotoxin, patulin. In this chapter, pre- and postharvest factors that affect the incidence of blue mold development are discussed. Synthetic fungicides provide control of blue mold disease and the resistance development in P. expansum to benzimidazole fungicides in the 1980s has led to the search for novel reduced-risk fungicides, biological control agents (BCA) and other control alternatives. To address the barriers that limit the efficacy of conventional and biological control methods, future research and implementation should focus on developing integrated disease management strategies that combine conventional and biological control systems with one or more of the physical, chemical, biological and genetic control methods, to provide effective management of postharvest blue mold. This chapter will examine the nature of the postharvest pathogen, P. expansum and the blue mold disease it causes in fruits and vegetables, factors affecting infection before harvest, at harvest and postharvest, and conventional and alternative methods for controlling the pathogen and the blue mold disease in storage. The literature search indicates that blue mold disease in apples and P. expansum have been thoroughly studied because of their economic importance in horticulture and the ability of some strains to produce the mycotoxin, patulin, that affects humans, however, other P. expansum and crop combinations have been less intensely studied.

Evaluation of fludioxonil for the control of post-harvest gray mould and blue mould in pears in cold and controlled atmosphere storages

Abstract Fludioxonil, a reduced-risk fungicide, was evaluated for the control of postharvest gray mould (caused by Botrytis cinerea) and blue mould (caused by Penicillium expansum) in pears in cold and controlled atmosphere (CA) storages. Lower blue mould incidence was observed in pears stored at 0°C as compared to pears stored at 4°C for up to 60 days, while lowest gray mould incidence was observed in both storages. In a time-course study, two of the highest concentrations tested, 300 and 600 µg mL−1, suppressed gray mould and blue mould for up to 100 days at 0°C and an increase in disease incidence was observed in the subsequent shelf-life studies. Both drench and dip application methods of fludioxonil at a concentration of 600 µg mL−1 provided effective control of gray mould and blue mould in CA storage for 4.5 months. Fludioxonil has the potential to be incorporated into the thiabendazole resistance management strategies for the control of postharvest diseases of pears.