Inhibitory effect of <Emphasis Type="Italic">Lycium europaeum</Emphasis> extracts on phytopathogenic soil-borne fungi and the reduction of late wilt in maize

The ability to control soil-borne pathogens in agriculture is highly conditioned by the restricted use of synthetic pesticides. Allelopathy, the antimicrobial activity of plant extracts, is a promising option against crop pathogens. Extracts from Lycium spp. such as L. barbarum, L. chinense and L. intricatum possess biological and therapeutic properties. Individual methanolic extracts from leaves and stems of the Mediterranean medicinal species L. europaeum collected in two locations of Tunisia were each evaluated in vitro against Verticillium dahliae (Vd), Sclerotinia sclerotiorum (Ss) and Harpophora maydis (Hm). The mycelial growth of the three fungi was significantly reduced by all the extracts at doses of 10 and 30 μl mL^?1 (equivalent to 1 and 3 mg plant tissue mL^?1). The sporulation of Hm was almost completely inhibited in all the amendments, but that of Vd was stimulated by one of the leaf extracts when 1 and 3 mg dried plant tissue mL^?1 were used. Sclerotia of Ss were formed in a smaller number, their total weight increasing at extract doses equivalent to 1 mg plant tissue mL^?1 and higher. In greenhouse, the pathogenicity of Hm was confirmed as early as 6 weeks after inoculation, since it caused significant decreases of weights in both roots and aboveground parts of maize. The detrimental effect of Hm on maize root weight in greenhouse was significantly counteracted by one of the leaf extracts added by watering. In total, 11 phenolic compounds were separated in the four extracts. The hydroxycinnamic acid family, including chlorogenic acid as a major compound, represented more than 50% of the total content in all the samples. Rutin was the most abundant flavonoid. The results of this work show the detrimental effect of L. europaeum extracts against the soil-borne pathogens Hm, Ss and Vd, and highlight their potential in crop protection if adequately developed into final products and used in combination with other tools.     

The application of lettuce in the qualitative and quantitative detection of <Emphasis Type="Italic">Rhizoctonia Solani</Emphasis> AG- 1 IA toxins

The toxins produced by Rhizoctonia solani are important causal agents of rice sheath blight. Effective detection of such toxins could improve the determination of the virulence of this agronomically important fungal pathogen. As such, the objective of the current study was to investigate the use of a variety of plant species [annual sowthistle (Sonchus oleraceus L.), Chinese cabbage (Brassica chinensis), spinach (Spinacia oleracea L.), lettuce (Lactuca sativa L. var. sativa), long leaf lettuce (Lactuca sativa var. ramosa Hort) and tobacco (Nicotiana tabacum)] for qualitative detection of R. solani crude toxins (RHCT) to replace the current rice leaf sheath based assay. This is constrained as rice plant takes long time to grow before the leaf sheath can be harvested From the initial screen, it was found that detached lettuce leaves provided the best alternative to rice material. Quantitative determination of RHCT activity by the phosphorus extravasation method was then performed on both rice (Oryza sativa L.) and lettuce. The results demonstrated that the detached lettuce leaves had the advantages of fast onset of symptoms, high sensitivity and non-perishability after inoculation. The quantity of phosphorus exosmosis observed in both lettuce leaves and rice leaf sheaths were significantly positively correlated. These data indicate that lettuce leaves can be used as a substitute material for rice leaf sheaths, with which to study the RHCT both qualitatively and quantitatively. The current study provides a new way to qualitatively and quantitatively detect RHCT.     

Integrative identification and molecular phylogeny of dagger and needle nematodes associated with cultivated olive in Tunisia

The occurrence and geographic distribution of longidorid nematode species inhabiting the rhizosphere of cultivated olive (cvs. Chemlali and Chétoui) in Tunisia were investigated. Morphological and morphometrical studies identified three Longidorus and six Xiphinema species, with frequencies of prevalence as following: Longidorus africanus (23.0 %), L. euonymus (4.5 %), L. glycines (13.7 %), Xiphinema conurum (13.7 %), X. italiae (36.4 %), X. meridianum (13.7 %), X. pachtaicum (18.2 %), X. robbinsi (9.1 %), and Xiphinema sp. (4.5 %). The three Longidorus species were reported for the first time in Tunisia, in addition to two species of Xiphinema (viz. X. meridianum and X. robbinsi). Molecular characterisation using D2-D3 expansion regions of 28S rRNA and ITS1-rRNA was carried out and Bayesian inference analysis was used to reconstruct phylogenetic relationships among these species and with other longidorids. Twenty-five new D2-D3 of 28S rRNA gene sequences were obtained in the present study, seven for Longidorus and 18 for Xiphinema spp., as well as 14 new ITS1 rRNA gene sequences (seven for Longidorus and seven for Xiphinema spp.).     

Insecticidal activity of plant-derived extracts against different economically important pest insects

With the aim of selecting potential botanical insecticides, seven plant extracts (Daphne mucronata (Family: Thymelaeaceae), Tagetes minuta (Asteraceae), Calotropis procera (Apocynaceae), Boenninghausenia albiflora (Rutaceae), Eucalyptus sideroxylon (Myrtaceae), Cinnamomum camphora (Lauraceae) and Isodon rugosus (Lamiaceae)) were screened for their toxic effects against four important agricultural pest insects, each representing a separate insect order; pea aphids of Acyrthosiphon pisum (Hemiptera), fruit flies of Drosophila melanogaster (Diptera), red flour beetles of Tribolium castaneum (Coleoptera), and armyworms of Spodoptera exigua (Lepidoptera). Aphids were the most susceptible insect with 100% mortality observed after 24 h for all the plant extracts tested. Further bioassays with lower concentrations of the plant extracts against aphids, revealed the extracts from I. rugosus (LC₅₀ 36 ppm and LC₉₀ 102 ppm) and D. mucronata (LC₅₀ 126 ppm and LC₉₀ 198 ppm) to be the most toxic to aphids. These most active plant extracts were further fractionated into different solvent fractions on polarity basis and their insecticidal activity evaluated. While all the fractions showed considerable mortality in aphids, the most active was the butanol fraction from I. rugosus with an LC₅₀ of 18 ppm and LC₉₀ of 48 ppm. Considering that high mortality was observed in aphids within 24 h of exposure to a very low concentration of the butanol fraction from I. rugosus, we believe this could be exploited and further developed as a potential plant-based insecticide against sucking insect pests, such as aphids.     

<Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> upregulates marker genes of the salicylic acid and ethylene signaling pathways but not those of the jasmonic acid pathway in leaflets of <Emphasis Type="Italic">Solanum</Emphasis> lines during early stage of infection

Real-Time PCR assay was used to quantify the expression of marker genes of the salicylic acid, jasmonic acid and ethylene signaling pathways in seven Solanum lines after inoculation with a Ralstonia solanacearum phylotype I strain, R008. Four Solanum lycopersicum lines (CRA 66, Hawaii 7996, MST 32/1, Quatre carrées), one S. tuberosum line (Spunta), the wild Lycopersicon cerasiforme and Solanum commersonii were used for this investigation. Results revealed very little activation of the jasmonic acid pathway marker genes, lipoxygenase A (LoxA) and protease inhibitor II (Pin2), with no significant difference (p > 0.05) in fold change expression among the Solanum lines. In contrast the salicylic acid pathway marker genes, glucanase A (GluA) and PR-1a, and the ethylene pathway marker genes, osmotin-like (Osm) and PR-1b, were expressed at higher levels with a statistically significant difference (p < 0.05) in fold change expression among the Solanum lines. The resistant lines L. cerasiforme, CRA 66, Hawaii 7996 and S. commersonii showed stronger activation of the salicylic acid and ethylene marker genes than the moderately resistant cultivar (MST 32/1) and the susceptible lines (Quatre carrées and Spunta). The marker genes reached their highest expression levels earlier (4 h.p.i) in the resistant and moderately resistant lines than in the susceptible lines (48 h.p.i.). These results indicate that salicylic acid and ethylene signaling pathways have a significant role in defense against R. solanacearum. The timing and magnitude of the upregulation of gene expression may determine the plant ability to put up a defense response against the pathogen.     

Control of infection of tomato fruits by <Emphasis Type="Italic">Alternaria</Emphasis> and mycotoxin production using plant extracts

Tomato fruits are susceptible to infection by Alternaria species. In addition, Alternaria species may contaminate the fruits with mycotoxins. There is thus interest in control systems to minimise pathogenicity and control toxin production. The objectives of this study were to examine the effect of plant extracts of Eucalyptus globulus and Calendula officinalis on the growth of strains of Alternaria alternata and A. arborescens, on pathogenicity of tomato fruits and mycotoxin production. The growth bioassays showed that the ethanolic and chloroformic fractions of E. globulus were the most effective in reducing growth of A. alternata (66–74 %) and A. arborescens (86–88 %), respectively at 2500 μg/g. The effects of plant extracts on mycotoxin biosynthesis were variable and strain dependent. The most effective fractions in decreasing mycotoxin accumulation were the ethanolic and chloroformic extracts of E. globulus, which reduced tenuazonic acid by 89 %, alternariol by 75–94 % and almost complete inhibition of alternariol monomethyl ether. All the tested fractions reduced percentage of infected tomato fruits when compared to the controls. The ethanolic and chloroformic fractions of E. globulus completely inhibited growth of A. alternata and A. arborescens on unwounded fruits and reduced the aggressiveness on wounded fruits of strains of both species significantly.     

Pathogenicity of <Emphasis Type="Italic">Plectosphaerella</Emphasis> species on lettuce and susceptibility of lettuce cultivars

Plectosphaerella rot affects hydroponically grown lettuce, especially those grown using deep flow technique. Plectosphaerella species such as P. pauciseptata and P. cucumerina are reported as causal agents of this disease. However, the relation between fungal lineage and pathogenicity on lettuce has been unclear. From inoculation tests, we discovered that various lineages of Plectosphaerella can infect lettuce tissues. Even strains isolated from non-lettuce plants were pathogenic on lettuce. Furthermore, various lettuce cultivars were equally susceptible to a particular strain. These results indicate that strains from a wide lineage of Plectosphaerella can be pathogenic on various lettuce cultivars.     

Conidial sporulation of <Emphasis Type="Italic">Stemphylium solani</Emphasis> under laboratory conditions and infectivity of the inoculum produced <Emphasis Type="Italic">in vitro</Emphasis>

Potato virus Y (PVY) is the type-species of the genus Potyvirus, family Potyviridae, being reported as a major tomato (Solanum lycopersicum L.) pathogen in several regions of the world. Pepper yellow mosaic virus (PepYMV) was originally described as a resistance-breaking Potato virus Y (PVY) isolate on Capsicum annuum L. cultivars, and afterwards it was also reported infecting tomatoes in Brazil. In the present work, a search for sources of resistance to both PepYMV and PVY was conducted in a collection of 119 accessions belonging to seven Solanum (section Lycopersicon) species. This germplasm was initially evaluated to PepYMV reaction by mechanical inoculation followed by symptom observations and ELISA. Potential PepYMV resistance sources were identified for the first time in S. habrochaites, S. peruvianum, S. corneliomuelleri, S. chilense, S. pimpinellifolium, and one accession derived from an interspecific cross (S. lycopersicum x S. peruvianum). A sub-group of 24 accessions with negative serology for PepYMV was also challenged with a PVY isolate, followed by serological and molecular detection with universal primers. Solanum habrochaites ‘L.03683’ and ‘L.03684’ were the only accessions found with stable resistance to both viruses. These results confirm S. habrochaites as the most important source of multiple resistance factor(s) to distinct Potyvirus species.     

Infection of <Emphasis Type="Italic">Sorghum bicolor</Emphasis>, selected grass species and <Emphasis Type="Italic">Zea mays</Emphasis> by G<Emphasis Type="Italic">loeocercospora sorghi</Emphasis>, causal pathogen of zonate leaf spot

Zonate leaf spot (Gloeocercospora sorghi) is a common disease in Sorghum bicolor producing areas of the U.S., but little is known about its biology, virulence and severity on S. bicolor, Zea mays, and related crop grassweeds. Greenhouse studies were conducted to determine and compare the virulence and severity of G. sorghi on 10 commercially available sorghum hybrids, four Z. mays hybrids and selected grassweed species including Sorghum bicolor (grain sorghum and shattercane biotypes) and Sorghum halepense (Johnsongrass), two of the most problematic arable weeds. Plants from the respective species were inoculated with a local G. sorghi isolate and maintained in a dew-chamber at 24 °C for 24 h and then incubated under greenhouse conditions for 4 weeks. Plants were observed for lesion expression and rated using a modified Horsfall-Barrett scale (0–10). The first symptoms of infection were visible within 24 h following inoculation on shattercane and S. bicolor hybrids. Symptoms consisted of small, non-diagnostic purple lesions on the leaves. Results showed that S. bicolor, S. halepense and shattercane were susceptible to G. sorghi. All other species tested in this study were not infected. More particularly, disease severity, increased from a rating of 3 to 10 on sorghum and from 2 to 7 on S. halepense between 2 and 23 days after inoculation, respectively. However, disease severity on shattercane increased rapidly from 3.5 to 10 between 2 and 8 days after inoculation, respectively. Among the sorghum hybrids tested, FFR-322 appeared to be the most resistant to G. sorghi while Pioneer 83G66 appeared to be the most susceptible. Z. mays hybrids were not infected by the fungus used in this study. G. sorghi could be used effectively to manage shattercane and S. halepense infestations occurring in Z. mays and S. bicolor fields consisting of specific G. sorghi-resistant hybrids.     

Foliar diseases of willows (<Emphasis Type="Italic">Salix</Emphasis> spp.) in selected locations of the Karkonosze Mts. (the Giant Mts)

The species of Salix herbacea L. and Salix lapponum L. are the rare relicts within the genus of Salix. With respect to their phylogeography, they are classified into the taxa of the Arctic-Alpine distribution. They can be found in the Arctic zone (Greenland, Scandinavia, Spitsbergen) as well as in such geographically separated areas as the mountains of lower latitudes like the Alps, the Sudetes or the Carpathians. Another species of willow occurring in the mountains of Europe, and more specifically on montane positions of the Balkan-Carpathian distribution is S. silesiaca. The aim of the study was to determine the severity of the diseases of the willows growing in selected locations in the Karkonosze Mountains. The research was conducted in the years 2014 and 2015 in the Kocio? ?omniczki [the ?omniczka Cirque] and the Wielki and Ma?y ?nie?ny Kocio?, [the Great and Small Snowy Cirques] at intervals of 4 weeks, from April to October. Field observations included the species of the genera Salix: S. herbacea, S. silesiaca and S. lapponum. Disease symptoms were identified with the percentage of infected plants at selected locations, as well as the percentage of infected leaves, recorded. In the course of the research, symptoms of rust caused by Melampsora epitea and M. alpina (S. herbacea and S. silesiaca), black spot blight caused by Rhytisma salicinum (S. silesiaca) and leaf spot, probably caused by a complex of fungal taxa (S. herbacea), were detected on the leaves of Salix spp. growing in post glacial cirques. During the study period, there were no disease symptoms on S. lapponum. A total of 13 species of fungi were isolated from the infected leaves of S. herbacea. In the first year, the highest share in the fungal assembly was taken by Penicillium notatum, followed by Alternaria alternata and an unidentified species of the genus Ceuthospora. In the second year of the research, Ceuthospora spp. and A. alternata predominated in the species assembly of fungi in the infected leaves of the herb willow.     

First report of sclerotinia rot of mango caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in Japan

A panicle blight with sclerotia was found on mango (Mangifera indica L.) in Okinawa Prefecture, Japan, in March 2016. Water-soaked lesions with white mycelia developed on panicles in the flowering stage; softening and decay of panicles was followed by formation of sclerotia. The fungus isolated from these sclerotia was identified as Sclerotinia sclerotiorum based on morphology and analysis of rDNA-ITS sequences. The isolate reproduced the symptoms on mango panicles in an inoculation test and was reisolated from flower stalks. This is the first report of sclerotinia rot (kinkaku-byo in Japanese) on mango caused by S. sclerotiorum in Japan.     

Mechanism of in vitro antagonism of phytopathogenic <Emphasis Type="Italic">Scelrotium rolfsii</Emphasis> by actinomycetes

Sclerotium rolfsii (Sr), a soil-borne fungal pathogen, causes disease in a wide range of crops. Recently, we identified five actinomycetes (Streptomyces globisporus subsp. globisporus, S. globisporus, S. flavotricini, S. pactum, and S. senoensis) showing significant inhibitory effects on plant pathogens. In this study, the effects of the five actinomycetes for the biocontrol of Sr were investigated using the plate culture method and microscopy examination. Two actinomycetes with higher inhibitory effect were subsequently examined for the inhibition of sclerotial germination of Sr in unsterile soil in vitro. The cell-free cultures of five actinomycetes mediated significant inhibition of hyphal growth and sclerotial formation and germination of Sr. All actinomycete strains exhibited the ability to produce extracellular cell wall degrading enzymes in the culture conditions. The crude enzyme suspensions of S. flavotricini and S. pactum hydrolyzed the cell wall of Sr. At a dose of 1 g per kilogram soil, the solid formulations of S. flavotricini and S. senoensis prevented germination of 24% and 68% of sclerotia, respectively. Our results provide evidence of effective strains for the biocontrol of Sr, in addition to a further understanding of the underlying mechanism.     

Search for reservoirs of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ and mollicutes in weeds associated with carrot and celery crops

Currently, the main arthropod vectored pathogens associated with carrot and celery crop diseases are ˋCandidatus Liberibacter solanacearum´, Spiroplasma citri and different phytoplasma species. Mitigation strategies require elucidating whether these pathogens survive in the weeds of these Apiaceae crops, which can act as reservoirs. Weed surveys were conducted in a vegetative cycle (April to October 2012) in the spontaneous vegetation that surrounded crops affected by ˋCa. L. solanacearum´, S. citri and/or phytoplasmas. Sixty-three species of 53 genera that belong to 23 botanical families were collected in the main carrot and celery Spanish production area. Species were identified, estimating coverage and abundance, and conserved in herbarium. Samples were analysed by nested-PCR with universal primers for phytoplasmas detection, and were sequenced for identification purposes; by conventional PCR for S. citri and real-time PCR for ˋCa. L. solanacearum´. The only detected pathogens were ˋCa. Phytoplasma trifolii´ (clover proliferation group 16Sr VI-A) in Amaranthus blitoides and Setaria adhaerens and ˋCa. P. solani´ (stolbur group 16Sr XII-A) in Convolvulus arvensis. These pathogens were also sporadically detected in celery or carrot crops. Unexpectedly, neither ˋCa. L. solanacearum´ nor S. citri was detected in the weed samples, despite the relatively high prevalence of these pathogens (less than 66 % and 25 %, respectively) in the surveyed plots. This suggests that weeds do not play an epidemiological role as reservoirs in the spread of such organisms in the studied region. The use of pathogen-free seed lots and the control of vectors are crucial for preventing the introduction and spread of these economical important pathogens to new areas.     

Insecticidal toxicity of thirteen commercial plant essential oils against <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae)

We evaluated the chemical composition of thirteen commercially available plant essential oils and their insecticidal activity against the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). Gas chromatography-mass spectrometry was used to characterize the chemical components of the essential oils. A total of 113 compounds were identified, with terpenes (>80%) and aromatic compounds as primary constituents. The toxicity of each pure essential oil was tested separately on third instar larvae and adult beet armyworms by topical application of 0.5 μl oil/ insect. All plant essential oils were found to be harmful to S. exigua, with third instar larvae showing significantly more susceptibility than adults. Essential oils of Cinnamomum zeylanicum and Juniperus virginiana showed the highest toxicity (mortality above 90%) to larvae, while C. zeylanicum and Pogostemon cablin oils were the most harmful compounds (95% mortality) to adults. Cymbopogon winterianus oil caused delayed mortality (similar to the effects of insect growth regulators) as well as malformations in pupae. C. winterianus, Ocimum basilicum and Rosmarinus officinalis oils significantly reduced fecundity, whereas no significant effects were observed on fertility.     

Efficacy of some rhizospheric and endophytic bacteria <Emphasis Type="Italic">in vitro</Emphasis> and as seed coating for the control of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> infecting durum wheat in Tunisia

Sixty two rhizospheric and endophytic bacterial strains were evaluated for their biocontrol effect on two aggressive Fusarium culmorum isolates (Fc2 and Fc3). We observed that 35 % and 23 % of the tested strains inhibited the in vitro growth of Fc2 and Fc3 respectively. The observed antagonism was due to inhibition by contact (13–19 % of the strains) or at distance (10–16 % of the strains) for both fungal isolates. Some of the antagonistic bacteria showed the ability to produce diffuse and/or volatile compounds that inhibit the growth, the sporulation and macroconidia germination of F. culmorum. None of the tested antagonistic bacteria showed chitinase activity on synthetic medium. The sequencing of the 16S rDNA genes of some antagonistic bacteria showed that they belong to the genera Bacillus, Pseudomonas and Microbacterium. The double inoculation of durum wheat seeds by the antagonistic bacterial strains (B13, B18, BSE1, BSE3 and B16E) and the two F. culmorum isolates showed that germination and seedling vigor were generally improved in vitro. The percentage of infected seeds was also reduced. In greenhouse trials, the biocontrol effectiveness of F. culmorum was dependant from the virulence of the fungal strain and the specificity of the antagonistic interaction between bacterial and fungal strains. The bacterial strains B18 and B16E reduced F. culmorum infection on durum wheat plants probably due to their antagonistic and plant growth promoting activities and they may be used in a mixture as seed biopriming inoculum for plant growth bio-promoting and Fusarium wheat diseases biocontrol.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus

Bradyrhizobium sp., a slow-growing nitrogen-fixing symbiotic bacterium of legumes and common root endophyte of other plants, is closely related to Candidatus Liberibacter asiaticus (Las), the uncultured putative pathogen associated with citrus huanglongbing (HLB). In attempts to isolate Las on a low-nutrient medium that had been used for the isolation of several uncultured bacteria of the alpha subclass of proteobacteria, slow-growing Bradyrhizobium spp. were isolated and identified by sequencing of 16S rDNA. The individual isolates tested weakly positive (Ct = 31.2–36.0) with the USDA primers commonly used in qPCR assays for Las in foliar tissues. Direct DNA extracts from roots of HLB symptomatic trees that contained sequences of Bradyrhizobium sp. had Ct values ranging from 31.2 to 36.5; sequences of Las were not present in those samples. Potential cross-reaction between DNA of members of the Rhizobiales and sequences amplified by the Las primers were tested in silico with the Primer-BLAST tool in NCBI. Similar to Las, Bradyrhizobium generated predicted 16S rDNA amplicon sizes of 78–79 bp with the qPCR primers and of 1167-1172 bp with the conventional PCR primers. Bradyrhizobium sequences of 16S rDNA had 1–7 mismatches and only 1 mismatch at the 3′ end of qPCR and conventional PCR primers confirming potential cross-reactivity. As Bradyrhizobium is usually not found in foliage, the USDA qPCR primers can be safely used to check leaves for the presence of Las, but a threshold value of 31.0 is recommended for Las detection in roots. Other primers should be tested for potential cross-reaction with members of the Rhizobiales.     

The potential of entomopathogenic fungi to control the lettuce root aphid,<Emphasis Type="Italic">pemphigus bursarius</Emphasis>

The lettuce root aphid,Pemphigus bursarius, is a commercially important pest of lettuce and occupies a niche amenable to the action of entomopathogenic fungi. As the first stage of a biological control programme, a bioassay was developed against adult apterousP. bursarius. An isolate ofMetarhizium flavoviride was pathogenic toP. bursarius and sporulated abundantly upon aphid cadavers. However, two isolates ofVerticillium lecanii, pathogenic against glasshouse whitefly and aphids, were only weakly pathogenic toP. bursarius.     

TaqMan PCR assay for detection and quantification of <Emphasis Type="Italic">Stagonosporopsis tanaceti</Emphasis> in pyrethrum seed and seedlings

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (C_t) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = ?0.999, P < 0.001) between the C_t value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices.     

Influence of exogenous cholesterol in Pythiaceae resistance to inhibition by <Emphasis Type="Italic">Trichoderma</Emphasis> antibiosis

The purpose of this study was to determine if exogenous cholesterol availability influenced Pythiaceae resistance to antibiosis. Characterisation of an isolate of Phytophthora erythroseptica and Pythium ultimum for tolerance to antibacterial compounds found that 0.05 g.l^?1 chloramphenicol inhibited mycelial growth by 96.6 % and 23.5 % respectively. However, the addition of cholesterol (0.01 g l^?1) to potato dextrose agar (PDA) containing 0.05 g l^?1 chloramphenicol was found to increase mycelial growth of P. erythroseptica, indicating a role for cholesterol in tolerance to inhibitory antibacterial compounds. To determine if this property extended to suppressive effects of a potential biocontrol agent, P. erythroseptica and P. ultimum were then tested against a cell-free filtrate of diffusible metabolites produced by a suppressive Trichoderma harzianum isolate in the presence and absence of cholesterol in PDA. In the absence of cholesterol, diffusible metabolites of the T. harzianum isolate were found to inhibit mycelial growth of P. erythroseptica and P. ultimum on PDA by 98 % and 63.6 % respectively (P?<?0.0001). However, the inhibitory effect of the metabolites was mitigated when 0.005 g l^?1 of cholesterol was present in PDA, with mycelial growth of P. ultimum and P. erythroseptica reduced by only 60.4 % and 41.8 %, respectively (P?<?0.0001), much less inhibition than was observed in the absence of cholesterol. These results demonstrated that access to exogenous cholesterol can influence the sensitivity of Pythiaceae species to antibiosis by positively influencing mycelial growth.