PRISE2: software for designing sequence-selective PCR primers and probes.

BackgroundPRISE2 is a new software tool for designing sequence-selective PCR primers and probes. To achieve high level of selectivity, PRISE2 allows the user to specify a collection of target sequences that the primers are supposed to amplify, as well as non-target sequences that should not be amplified. The program emphasizes primer selectivity on the 3' end, which is crucial for selective amplification of conserved sequences such as rRNA genes. In PRISE2, users can specify desired properties of primers, including length, GC content, and others. They can interactively manipulate the list of candidate primers, to choose primer pairs that are best suited for their needs. A similar process is used to add probes to selected primer pairs. More advanced features include, for example, the capability to define a custom mismatch penalty function. PRISE2 is equipped with a graphical, user-friendly interface, and it runs on Windows, Macintosh or Linux machines.ResultsPRISE2 has been tested on two very similar strains of the fungus Dactylella oviparasitica, and it was able to create highly selective primers and probes for each of them, demonstrating the ability to create useful sequence-selective assays.ConclusionsPRISE2 is a user-friendly, interactive software package that can be used to design high-quality selective primers for PCR experiments. In addition to choosing primers, users have an option to add a probe to any selected primer pair, enabling design of Taqman and other primer-probe based assays. PRISE2 can also be used to design probes for FISH and other hybridization-based assays

Investigations into Peach Replant Disease and Nematophagous Fungi

The research described in this dissertation examined peach replant disease and two nematode biological control systems using traditional plant pathology methods and molecular microbial ecology methods. In Chapter 1, the experiments identified microbes associated with peach replant disease in soils with various levels of disease symptoms. To identify bacteria, fungi and oomycetes associated with the replant disease, culture and culture-independent analyses were performed on DNA extracted from plant roots. Among the most abundant bacterial operational taxonomic units, 27 were negatively correlated with peach top weights while 10 were positively correlated. Among the most abundant fungi and oomycetes, negative and positive associations were identified between P. vexans and Trichoderma spp. and peach top weights, respectively, and verified with sequence-selective quantitative PCR analyses. In Chapter 2, the population dynamics between Dactylella oviparasitica and Heterodera schachtii were investigated. Higher initial D. oviparasitica populations were associated with lower final H. schachtii populations. Regression models showed that the initial densities of D. oviparasitica were only significant when predicting the final densities of H. schachtii J2 and eggs as well as fungal egg parasitism, while the initial densities of J2 were significant for all final H. schachtii measurements. H. schachtii-associated D. oviparasitica populations were greatly reduced in nematodes collected from soil compared to nematodes collected from roots. Finally, phylogenetic analysis of rRNA genes suggested that D. oviparasitica belongs to a clade of nematophagous fungi with a large geographical distribution. In Chapter 3, three strains of Pochonia chlamydosporia var. chlamydosporia were genetically characterized and examined for their biocontrol efficacies against Meloidogyne incognita. All strains exhibited different patterns with the enterobacterial repetitive intergenic consensus (ERIC) PCR analysis. Strains 1 and 4 were similar in the PCR analyses of ß-tubulin and the rRNA internal transcribed spacer. In greenhouse trials, all strains reduced the numbers of nematode egg masses. Strain 4 reduced almost 50% of the eggs, and reduced the numbers of J2 and root-galling. A newly developed small subunit rRNA-based PCR analysis differentiated strain 4 from the others, and could potentially be used as a screening tool for identifying other effective biocontrol strains of P. chlamydosporia var. chlamydosporia

The Evaluation of Egg-Parasitic Fungi Paraboeremia taiwanensis and Samsoniella sp. for the Biological Control of Meloidogyne enterolobii on Chinese Cabbage.

Meloidogyne enterolobii, an aggressive plant-parasitic nematode, has been causing great yield loss worldwide in recent years. With no resistant Chinese cabbage cultivar available currently, a biological control strategy is needed to offer an eco-friendly option for sustainable farming. In this study, the nematode suppression efficacy of two newly isolated fungi, Paraboeremia taiwanensis and Samsoniella sp., were evaluated against M. enterolobii and compared to the known biological control agents Hyalorbilia oviparasitica strain DoUCR50 and Purpureocillium lilacinum strain 251 (PL251). Both P. taiwanensis and Samsoniella sp. reduced 29%-63% disease severity as effectively as the commercial product PL251 on Chinese cabbage in greenhouse trails. The in vitro egg infection rate was 47.83% by P. taiwanensis and 47.50% for Samsoniella sp., respectively. A special protocol for scanning electron microscope observation of the fungi-infected nematodes was established in this study, and the egg parasitism of the four fungi against M. enterolobii was further confirmed. For all fungi examined in this study, fungal hyphae were seen apparently penetrating into M. enterolobii eggs without destructive damage of the overall outer eggshell and the hyphae continued to grow within eggs after 6 days of infection. The results of this study imply a similar egg-parasitism mechanism for P. taiwanensis, Samsoniella sp., H. oviparasitica DoUCR50, and P. lilacinum PL251. It further enlightens the application potential of nematophagous fungi as biocontrol agents against plant-parasitic nematodes in vegetable crop management

Fungi associated with the potato taste defect in coffee beans from Rwanda.

BackgroundPotato taste defect (PTD) of coffee is characterized by a raw potato like smell that leads to a lower quality taste in the brewed coffee, and harms the commercial value of some East African coffees. Although several causes for PTD have been proposed, none of them have been confirmed. Recently, high throughput sequencing techniques and bioinformatic analysis have shown great potential for identifying putative causal agents of plant diseases. Toward the goal of determining the cause of PTD, we examined raw coffee beans from Rwanda exhibiting varying PTD scores using an Illumina-based sequence analysis of the fungal rRNA ITS region.ResultsSix fungal amplicon sequence variants (ASVs) with high relative abundances correlated with coffee taste scores. Four of these ASVs exhibited negative correlations - Aspergillus versicolor, Penicillium cinnamopurpureum, Talaromyces radicus, and Thermomyces lanuginosus - indicating that they might be causing PTD. Two of these fungi exhibited positive correlations - Kazachstania humilis and Clavispora lusitaniae - indicating that they might be inhibiting organisms that cause PTD.ConclusionsThis study addressed PTD causality from a new angle by examining fungi with high throughput sequencing. To our knowledge, this is the first study characterizing fungi associated with PTD, providing candidates for both causality and biocontrol

Copy Number Quantification for the Soybean Cyst Nematode Resistance Locus rhg1 in the Soybean Varieties of Taiwan

Disease resistance is one of the most successful strategies in crop protection. For example, the implementation of PI 88788 type resistance, which contains high copy numbers of Resistance to Heterodera glycines 1 (rhg1) loci, into the commercial soybean varieties of the United States has significantly reduced the yield losses caused by soybean cyst nematode (SCN, H. glycines). Vegetable soybean, or edamame, has become a major exporting agricultural product in Taiwan with an annual revenue over $80 million USD since 2017. Several local varieties have been developed to fulfill the market needs such as the traits of flavor and sweetness. However, it remains unclear if the historical breeding programs ever incorporated rhg1 resistance into the varieties of Taiwan. This study applied the TaqMan qPCR method to measure the fluorescent signals specific to the rhg1 locus on the chromosome 18 of soybean, and the ratio of VIC and FAM signals were analyzed to predict the rhg1 copy number in the 21 soybean varieties of Taiwan. The results indicated the copy number and the single nucleotide polymorphisms of the 21 soybean varieties were identical to the susceptible soybean variety ‘Williams 82’. As importation of soybean will be continuously needed to fulfill the market and because SCN is absent in the soybean fields of Taiwan, lacking rhg1 resistance in the local soybean varieties may put the edamame industry at risk and early implementation of SCN resistance in the breeding program, alongside the application of quarantine regulations, will be the key to maintain the SCN-free status and to sustain the edamame industry in Taiwan

Correlations between Root-Associated Microorganisms and Peach Replant Disease Symptoms in a California Soil

<div><h3>Background</h3><p>Replant disease often occurs when certain crops are “replanted” in a soil that had previously supported the same or similar plant species. This disease typically leads to reductions in plant growth, crop yields, and production duration, and its etiology remains ill-defined. The objective of this study was to identify microorganisms associated with peach replant disease symptoms at a field location in California, USA. Soil samples were subjected to treatments to create various levels of replant disease symptoms. Clonal peach seedlings were grown in the treated soils in greenhouse trials. After 6 weeks, plant growth parameters were measured, and both culture and culture-independent analyses were performed to identify root-associated bacteria, fungi and stramenopiles.</p> <h3>Results</h3><p>A total of 295,785 bacterial operational taxonomic units (OTU) were identified by an Illumina-based, high throughput sequence analysis of rRNA genes. Among the 60 most abundant OTUs, 27 showed significant (<em>P</em><0.05) negative correlation with peach shoot weights while 10 were positively correlated. Most of these OTUs belonged to the bacterial phylum Proteobacteria (96%), including the classes Gammaproteobacteria (44.4%), Betaproteobacteria (33.3%) and Alphaproteobacteria (22.2%), and the orders Pseudomonadales, Burkholderiales, Chromatiales, Rhodocyclales, and Sphingomonadales. The most abundant fungi were <em>Trichoderma asperellum</em>, <em>Trichoderma virens, Fusarium oxysporum</em>, <em>Ceratocystis fimbriata</em> and <em>Fusarium solani</em>. The most abundant stramenopiles were <em>Pythium vexans</em>, <em>Pythium violae</em> and an unidentified <em>Aplanochytrium</em> species. Validation experiments using sequence-selective quantitative PCR analyses identified negative and positive associations between <em>P. vexans</em> and <em>Trichoderma</em> spp. and peach shoot weights, respectively.</p> <h3>Conclusions</h3><p>This study identified numerous microorganisms associated with peach replant symptoms, some of which have been previously identified while others represent new candidates. Subsequent Koch's postulates investigations will assess their possible roles in this replant disease.</p> </div

Relationships between bacteria and fresh peach shoot weights.

<p>A. <i>Pseudomonas fluorescens</i>. B. <i>Rhodanobacter lindaniclasticus</i>. Regression equations are (A) [log₁₀ reads per sample = 4.02−0.146 (grams of shoots); <i>P</i> = 0.015; R² = 18.3%, n = 32] and (B) [log₁₀ reads per sample = 2.59+0.069 (grams of shoots); <i>P</i> = 0.004; R² = 25.0%, n = 32]. Lines are from regression analyses.</p

Relative abundance of stramenopiles from peach seedling roots grown in soil exhibiting peach replant disease symptoms.

<p>A. Cultured isolates; values are % of 26 isolates. B. Culture-independent analysis; values are % of 48 sequences. Taxa without numbers have relative abundance values of less than 5%.</p