An ethnopharmacological approach to multidrug -resistant Staphylococcus aureus: Evaluation of Italian plants used in the traditional healing of skin disease

One-third of botanical remedies from southern Italy are used to treat skin and soft tissue infections (SST's). Methicillin-resistant Staphylococcus aureus (MRSA), a common cause of SSTIs, is responsible for increased morbidity and mortality from infections. Therapeutic options are limited by antibiotic resistance. Many plants possess potent antimicrobial compounds for these disorders. Validation of traditional medical practices is important for the people who rely on medicinal plants. Moreover, identification of novel antibiotics and anti-pathogenic agents for MRSA is important to global healthcare.^ I took an ethnopharmacological approach to understand how Italian medicinal plants used for the treatment of SSTIs affect MRSA growth and virulence. My hypothesis was that plants used in folk remedies for SSTI would exhibit lower cytotoxicity and greater inhibition of bacterial growth, biofilm formation and toxin production in MRSA than plants used for remedies unrelated to the skin or for plants with no ethnomedical application. The field portion of my research was conducted in the Vulture-Alto Bradano area of southern Italy. I collected 104 plant species and created 168 crude extracts. In the lab, I screened samples for activity against MRSA in a battery of bioassays. Growth inhibition was analyzed using broth microtiter assays for determination of the minimum inhibitory concentration. Interference with quorum-sensing (QS) processes, which mediate pathogenicity, was quantified through RP-HPLC of δ-toxin production. Interference with biofilm formation and adherence was assessed using staining methods. The mammalian cytotoxicity of natural products was analyzed using MTT cell proliferation assay techniques.^ Although bacteriostatic activity was limited, extracts from six plants used in Italian folk medicine (Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa, Marrubium vulgare, and Rubus ulmifolius ) significantly inhibited biofilm formation and adherence. Moreover, plants used to treat SSTI demonstrated significantly greater anti-biofilm activity when compared to plants with no ethnomedical application. QSI activity was evident in 90% of the extracts tested and extracts from four plants ( Ballota nigra, Castanea saliva, Rosmarinus officinalis, and Sambucus ebulus) exhibited a significant dose-dependent response. Some of the plant remedies for SSTI identified in this study can be validated due to anti-MRSA activity.^

Diffusible Signal Factor (DSF)-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell-to-cell signals of the Diffusible Signal Factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signaling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.

Monitoring Infection Risk for Air and Soil Borne Fungal Plant Pathogens using Antibody and DNA Techniques and Mathematical Models describing Environmental Parameters

Economic losses resulting from disease development can be reduced by accurate and early detection of plant pathogens. Early detection can provide the grower with useful information on optimal crop rotation patterns, varietal selections, appropriate control measures, harvest date and post harvest handling. Classical methods for the isolation of pathogens are commonly used only after disease symptoms. This frequently results in a delay in application of control measures at potentially important periods in crop production. This paper describes the application of both antibody and DNA based systems to monitor infection risk of air and soil borne fungal pathogens and the use of this information with mathematical models describing risk of disease associated with environmental parameters.

Genetic Modification of Disease Resistance: Fungal and Oomycete Pathogens

Development of recombinant DNA technology allowed scientists to manipulate plant genomes, making it possible to study genes and exploit them to modify novel agronomic traits. Here, we review the current and future potential of genetic modification (GM) strategies used to increase the resistance of plants to oomycete and fungal pathogens. Numerous resistance genes (R-genes) have been cloned, and under laboratory conditions, transgenic plants have given promising results against some important plant pathogens. However, only a few have so far been deployed as commercial crop plants.GMof plants to disrupt pathogenicity, such as by inhibiting or degrading pathogenicity factors, especially by necrotrophic pathogens, has also been exploited. The potential to engineer plants for the production of antimicrobial peptides or to modify defense-signaling pathways have been successfully demonstrated under laboratory conditions. The most promising current technology is genome editing, which allows researchers to edit DNA sequences directly in their endogenous environment. The potential of this approach is discussed in detail and examples where broad-spectrum resistance has been achieved are given.

Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources

QTL identified for seedling and adult plant crown rot resistance in four partially resistant hexaploid wheat sources. PCR-based markers identified for use in marker-assisted selection. Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in many wheat-growing regions globally. Complete resistance to infection by F. pseudograminearum has not been observed in a wheat host, but germplasm with partial resistance to this pathogen has been identified. The partially resistant wheat hexaploid germplasm sources 2-49, Sunco, IRN497 and CPI133817 were investigated in both seedling and adult plant field trials to identify markers associated with the resistance which could be used in marker-assisted selection programs. Thirteen different quantitative trait loci (QTL) conditioning crown rot resistance were identified in the four different sources. Some QTL were only observed in seedling trials whereas others appeared to be adult plant specific. For example while the QTL on chromosomes 1AS, 1BS, and 4BS contributed by 2-49 and on 2BS contributed by Sunco were detected in both seedling and field trials, the QTL on 1DL present in 2-49 and the QTL on 3BL in IRN497 were only detected in seedling trials. Genetic correlations between field trials of the same population were strong, as were correlations between seedling trials of the same population. Low to moderate correlations were observed between seedling and field trials. Flanking markers, most of which are less than 10 cM apart, have now been identified for each of the regions associated with crown rot resistance.

Optimising plant bed performance through adaptive research with the Australian sweetpotato industry

A key driver of Australian sweetpotato productivity improvements and consumer demand has been industry adoption of disease-free planting material systems. On a farm isolated from main Australian sweetpotato areas, virus-free germplasm is annually multiplied, with subsequent 'pathogen-tested' (PT) sweetpotato roots shipped to commercial Australian sweetpotato growers. They in turn plant their PT roots into specially designated plant beds, commencing in late winter. From these beds, they cut sprouts as the basis for their commercial fields. Along with other intense agronomic practices, this system enables Australian producers to achieve worldRSQUOs highest commercial yields (per hectare) of premium sweetpotatoes. Their industry organisation, ASPG (Australian Sweetpotato Growers Inc.), has identified productivity of mother plant beds as a key driver of crop performance. Growers and scientists are currently collaborating to investigate issues such as catastrophic plant beds losses; optimisation of irrigation and nutrient addition; rapidity and uniformity of initial plant bed harvests; optimal plant bed harvest techniques; virus re-infection of plant beds; and practical longevity of plant beds. A survey of 50 sweetpotato growers in Queensland and New South Wales identified a substantial diversity in current plant bed systems, apparently influenced by growing district, scale of operation, time of planting, and machinery/labour availability. Growers identified key areas for plant bed research as: optimising the size and grading specifications of PT roots supplied for the plant beds; change in sprout density, vigour and performance through sequential cuttings of the plant bed; optimal height above ground level to cut sprouts to maximise commercial crop and plant bed performance; and use of structures and soil amendments in plant bed systems. Our ongoing multi-disciplinary research program integrates detailed agronomic experiments, grower adaptive learning sites, product quality and consumer research, to enhance industry capacity for inspired innovation and commercial, sustainable practice change.

The importance of cotton disease surveys in Queensland for monitoring endemic diseases and detecting new pathogens and pests

The cotton industry in Australia funds biannual disease surveys conducted by plant pathologists. The objective of these surveys is to monitor the distribution and importance of key endemic pests and record the presence or absence of new or exotic diseases. Surveys have been conducted in Queensland since 2002/03, with surveillance undertaken by experienced plant pathologists. Monitoring of endemic diseases indicates the impact of farming practices on disease incidence and severity. The information collected gives direction to cotton disease research. Routine diagnostics has provided early detection of new disease problems which include 1) the identification of Nematospora coryli, a pathogenic yeast associated with seed and internal boll rot; and 2) Rotylenchulus reniformis, a plant-parasitic nematode. This finding established the need for an intensive survey of the Theodore district revealing that reniform was prevalent across the district at populations causing up to 30% yield loss. Surveys have identified an exotic defoliating strain (VCG 1A) and non-defoliating strains of Verticillium dahliae, which cause Verticillium wilt. An intensive study of the diversity of V. dahliae and the impact these strains have on cotton are underway. Results demonstrate the necessity of general multi-pest surveillance systems in broad acre agriculture in providing (1) an ongoing evaluation of current integrated disease management practices and (2) early detection for a suite of exotic pests and previously unknown pests.

Production locality influences postharvest disease development and quality in mangoes

Postharvest disease management is one of the key challenges in commercial mango supply chains. Comprehensive investigations were made regarding the impact of geographic locality on postharvest disease development and other quality parameters in 'Sindhri' and 'Samar Bahisht (S.B.) Chaunsa' mangoes under ambient (33±1°C; 55-60% RH) and low temperature storage/simulated shipping (12±1°C; 80- 85% RH) conditions (28 or 35 days storage for 'Sindhri' and 21 or 28 days for 'S.B. Chaunsa'). Physiologically mature (days from fruit set were 95-100 and 110-115 for 'Sindhri' and 'S.B Chaunsa', respectively) 'Sindhri' and 'S.B. Chaunsa' fruits were harvested from five geographic localities and subjected to ambient and simulated shipping conditions. Under ambient conditions, no disease incidence was observed till fruit eating stage in 'Sindhri'. However, in 'S.B. Chaunsa', significant variation in different localities was observed with respect to disease incidence. Maximum and at par disease was exhibited by the fruit collected from district Vehari and Khanewal in 'S.B. Chaunsa'. Under simulated shipping conditions, disease development varied significantly with respect to different locations and storage durations. In 'Sindhri', fruit of M. Garh, while, 'S.B. Chaunsa' fruit of districts R.Y. Khan, M. Garh and Khanewal showed higher disease incidence. Fruit peel colour development was significantly reduced as storage days increased. Fruit firmness, skin shriveling, fresh weight loss, dry matter, biochemical and organoleptic attributes also varied significantly among the fruit sourced from different orchards of different localities. Analysis of N contents in leaves and fruit peel revealed that N contents of leaf and peel were positively correlated with disease severity in mango. Botryodiplodia spp., Phomopsis mangiferae, Alternaria alternata, Colletotrichum gloeosporioides were the pathogens isolated from fruits of all locations; however, the prevalence frequency varied with the geographic localities. In conclusion, the production locality, cultivar and nutrition (nitrogen content of fruit peel) had significant effect on fruit quality out-turn at ripe stage in terms of disease development so area specific disease management system needs to be implemented for better quality at retail.

An assessment of Pythium spp. associated with soft rot disease of ginger (Zingiber officinale) in Queensland, Australia

In Australia, Pythium soft rot (PSR) outbreaks caused by P. myriotylum were reported in 2009 and since then this disease has remained as a major concern for the ginger industry. From 2012 to 2015, a number of Pythium spp. were isolated from ginger rhizomes and soil from farms affected by PSR disease and assessed for their pathogenicity on ginger. In this study, 11 distinct Pythium spp. were recovered from ginger farms in Queensland, Australia and species identification and confirmation were based on morphology, growth rate and ITS sequences. These Pythium spp. when tested showed different levels of aggressiveness on excised ginger rhizome. P. aphanidemartum, P. deliense, P. myriotylum, P. splendens, P. spinosum and P. ultimum were the most pathogenic when assessed in vitro on an array of plant species. However, P. myriotylum was the only pathogen, which was capable of inducing PSR symptoms on ginger at a temperature range from 20 to 35 °C. Whereas, P. aphanidermatum only attacked and induced PSR on ginger at 30 to 35 °C in pot trials. This is the first report of P. aphanidermatum inducing PSR of ginger in Australia at high temperatures. Only P. oligandrum and P. perplexum, which had been recovered only from soils and not plant tissue, appeared non-pathogenic in all assays.

Accumulation, persistence and effects of indospicine residues in camels fed Indigofera plant

Indospicine (L-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as I. linnaei and I. spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution and histopathological effects of dietary indospicine on camels. Six young camels (2 – 4 year old), weighing 270 − 390 kg were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw); followed by the muscle, heart and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining 3 camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated mono-exponential decreases.

Mealybug wilt disease

Mealybug wilt disease (MWD) is a serious field disease of pineapples worldwide that was first described in Hawaii in 1910. MWD is thought to be caused by a complex involving viruses, mealybugs and ants. The viruses are transmitted by mealybugs, which in turn are tended by ants. Although a number of distinct viruses have been associated with the disease, the identity of the causal agent(s) has not been determined unequivocally. This chapter describes the disease symptopms, aetiology and management of MWD. In the last 20 years, significant advances have been achieved in identifying the causal viral agents, and gaining a better understanding of MWD. However, the interactions between the viruses, mealybugs and environmental factors are complicated, and the conditions required for the expression of MWD have only been partially elucidated at this time. The possible role of gene silencing, the identity of the additional ampelovirus(es) and badnavirus(es) that have been detected but not characterized, and the interaction between these disease-inducing factors are fertile areas for future research.

Rootstock selection, nitrogen and calcium influence postharvest disease in avocado

Field trials evaluating several parameters of growth, fruit yield and quality of 'Hass' avocado grafted to different rootstocks were established in 2004-2005 in four different growing regions of Australia. Fruit were harvested in three seasons from 2008, ripened and assessed for severity and incidence of anthracnose and stem end rot diseases. Peel samples were collected at harvest and analysed for concentrations of the cations (N, K, Ca, Mg). Rootstock significantly affected marketability of fruit (no stem end rot and less than 5% anthracnose) in 58% of the total number of trials evaluated, with better quality fruit harvested from 'Hass' grafted to Guatemalan or West Indian rootstocks such as 'A10' or 'Velvick'. Fruit quality was frequently poor from trees grafted to Mexican race rootstocks, regardless of growing location. Correlation analyses showed that fruit from rootstocks with superior fruit quality was often associated with lower skin N and higher Ca concentrations. There were significant positive correlations between anthracnose and skin N or N:Ca ratio in 75% of trials evaluated. There was a significant negative correlation between anthracnose and Ca in 42% of trials. The correlations between stem end rot and skin N (positive) or Ca (negative) were each significant in 42% of trials. Based on the results in this project, N:Ca ratios in the skin of unripe avocado fruit at harvest may provide one of the best indicators of potential postharvest disease in ripe fruit, and may have implications for fertiliser regimes.

HERITABILITY FOR RESISTANCE TO ROSETTE DISEASE IN EXOTIC VALENCIA GROUNDNUTS

Groundnut rosette disease (GRD) is the most destructive virus disease of Valencia groundnuts ( Arachis hypogaea L.) in sub-Saharan Africa. Cultural, biological and chemical control measures have received limited success due to small scale farmers’ inability to use them. Use of host plant resistance provides the most effective and economically viable management option for the resource poor farmers. This study was conducted to determine heritability for resistance to GRD in Valencia groundnuts. Six crosses; Valencia C (P1) × ICGV-SM 90704 (P2), Valencia C (P1) × ICGV-SM 96801(P2), Valencia C (P1) × ICGV-SM 99566 (P2), NuMex-M3 (P1) × ICGV-SM 90704 (P2), NuMex-M3 × ICGV-SM 96801 (P2), and NuMex-M3 (P1) × ICGV-SM 99566 (P2), were made to generate F1, F2, BC1P1 and BC1P2 populations. Data on GRD severity were collected on a 1-9 score scale. Genetic Advance as a percentage of the mean (GAM) and heritability were estimated using variance components. Phenotypic Coefficient of Variation (PCV) and Genotypic Coefficient of Variation (GCV) estimates were high (20.04-70.1%) in the six crosses, except for Valencia C × ICGV-SM 96801(18.1%) and NuMex-M3 × ICGV-SM 96801(17.1%), which exhibited moderate GCV values. Broad and narrow sense heritability estimates for GRD disease score ranged from 64.1 to 73.7% and 31 to 41.9%, respectively, in all the crosses. GAM was high in all the crosses (21-50.7%), except for Valencia C x ICGV-SM 96801 (14.67), M3 x ICGV-SM 99566 (18%) and NuMex-M3 x ICGV-SM 96801 (13.5%) crosses that exhibited moderate GAM. The study revealed the presence of variability of GRD resistance, implying that genetic improvement of these exotic materials is possible.

In vitro technique for selecting onion FOR white rot disease- resistance

In vitro selection is one of the most effective and efficient techniques for plant improvement. This is due to its ability to isolate plants with the desired character(s), either by applying a selection agent on the culture media to drive the selection of somaclones with the required character(s), or by establishing particular conditions that change in the genomes of somaclones toward the required character. The objective of this study was to identify a suitable protocol for in vitro selection of Allium white rot disease ( Sclerotium cepivorum ) tolerance in commercial Egyptian onion varieties, namely Giza 20, Giza 6 and Beheri Red. Oxalic acid (OA), the phytotoxin produced by Sclerotium cepivorum, was used as the selective agent. Seeds of the three Egyptian varieties were germinated on four concentrations (0.0, 0.02, 0.2, 2 and 20 mM) of Oxalic acid. Among the tested cultivars, Beheri Red had the highest germination frequency (52%) at all concentrations tested, followed by Giza 20 (42.6%), and Giza 6 at (32%). Cotyledon explants from the varieties were cultured on toxic MSBDK medium, supplemented with 0, 3, 6 and 12 mM OA. The survival of calli on MSBDK free toxic medium was 70.7% for all tested cultivars; however, MSBDK-stressed medium, with 3 mM OA reduced the viable calli to 42.1%. The highest OA concentration (12 mM) completely inhibited calli induction from cotyledons explants. A medium supplement with 3 mM OA retarded 80% of calli growth. Among 156 tested calli of Beheri Red, only 23 calli (14.7%) survived on toxic medium for 45 days. Similarly, there was 15.6% survival for Giza 20 calli, while 40.1% of the Giza 6 calli survived. Plantlets were regenerated from surviving calli and transplanted onto ex vitro, and formed bulb after acclimatisation.

A bacterial acetyltransferase targets the protein kinase ZIP1, a positive regulator of plant immunity

Pseudomonas syringae is a model bacterial pathogen that penetrates the leaf to reach the plant apoplast, where it replicates causing disease. In order to do that, the pathogen must interfere and suppress a two-tiered plant defense response: PTI (PAMP-Triggered Immunity, or basal resistance) and ETI (Effector-Triggered Immunity). P. syringae uses a type III secretion system to directly deliver effector proteins inside the plant cell cytosol, many of which are known to suppress PTI, some of which are known to trigger ETI, and a handful of which are known to suppress ETI. Bacterial infection can also trigger a systemic plant defense response that protects the plant against additional pathogen attacks known as SAR (Systemic Acquired Resistance). We are particularly interested in the molecular and cellular mechanisms involved in effector-mediated defense evasion by P. syringae, in particular those involved in the suppression of ETI and SAR, and/or mediation of hormone signaling. Here we present data describing effector-mediated interference with plant immunity, by means of acetylation of a key positive regulator of local and systemic responses. Our work identifies a novel plant target for effector function, and characterizes its function. This work illustrates how analyzing the means by which a given effector interferes with its target can provide novel information regarding eukaryotic molecular mechanisms.