Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot

Background Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. Methods We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. Results P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. Conclusions P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Timber Losses during Harvesting in Managed Shorea robusta Forests of Nepal

Logging and sawing of timber using conventional tools by unskilled workers causes enormous damage to the valuable timber, residual stand, regeneration, and forest soil in Nepal. The purpose of this study was to find out the volume reduction factor and identify major strategies to reduce timber losses in the tree harvesting process in the Terai Shorea robusta forest of Nepal. Field measurements and product flow analysis of 51 felled trees from felling coupes and randomly selected 167 sawed logs were examined to study harvesting losses. Responses from 116 forest experts were analyzed to explore strategies for reducing harvesting and processing losses. The results showed that timber losses in the felling and bucking stage with and without stem rot were 23% and 22%, respectively. Similarly, timber losses in the sawing stage with and without stem rot were 31% and 30%, respectively. Paired t-test at 5% level of significance revealed that there was significant loss in both tree felling and log sawing stages with present harvesting practice. The most leading factor contributing to timber loss in all of the three stages was the use of inappropriate equipment during tree harvesting. Use of synthetic ropes for directional felling and skidding as well as flexible and portable sawing machine with size adjustment options during sawing were mainly recommended as strategies to reduce timber losses. This study serves as a baseline study to identify and quantify timber losses in different stages of tree conversion and also formulate their reduction strategies in Nepal.

Formula padat Bacillus cereus STRAIN TLE1.1 untuk pengendalian penyakit busuk pangkal batang (Sclerotium rolfsii) pada tanaman tomat

Penyakit utama tanaman tomat yaitu busuk pangkal batang yang disebabkan oleh Sclerotium rolfsii dapat menimbulkan kerugian mencapai 80-100%. Tujuan penelitian yaitu untuk mendapatkan formula padat Bacillus cereus strain TLE1.1 yang efektif untuk pengendalian penyakit busuk pangkal batang pada tanaman tomat. Penelitian bersifat eksperimen dengan mengamati kemampuan formula padat B. cereus strain TLE1.1 dalam pengendalian penyakit busuk pangkal batang dengan Rancangan Acak Lengkap yang terdiri atas 9 perlakuan dan 3 ulangan. Perlakuan terdiri atas kombinasi bahan pembawa formula padat yang terdiri atas limbah padat ampas tebu, ampas tahu dan tongkol jagung, fungisida serta kontrol. Masing-masing formula padat B. cereus strain TLE1.1 diintroduksi pada benih dan bibit tomat. Hasil penelitian menunjukkan bahwa hampir semua formula mampu menekan penyakit busuk pangkal batang tanaman tomat. Formula terbaik dalam menurunkan penyakit busuk pangkal batang pada tanaman yaitu formula ampas tahu dan ampas tahu + tongkol jagung.Main disease of tomato plant, namely stem rot caused by Sclerotium rolfsii which can cause losses up to 80-100%. The aim of the study was to obtain a solid formula of Bacillus cereus strain TLE1.1 which was effective for controlling stem rot disease in tomato plant. This research was an experimental study to know the ability of the solid formula of B. cereus strain TLE1.1 in controlling stem rot disease which was carried out in a completely randomizeddesign consisting of 9 treatments and 3 replications. The treatment consistedof a combination of solid formula carriers consisting of sugarcane solid waste,tofu dreg and corncob, fungicides and controls. Each solid formula of B. cereus strain TLE1.1 was introduced into tomato seeds and seedlings. The results showed that almost all of the formulas were able to suppress stem base disease of tomato plants. The best formula that reduced stem rot in plants were the tofu dreg and tofu dreg + corncob formula.

Microclimate Modification through Groundnut-Pigeon Pea Intercropping System and its Effect on Physiological Responses, Disease Incidence and Productivity

Background: Groundnut (Arachis hypogaea) is the predominant leguminous oilseed crop of India which has turned out to be a sensitive victim to climate change episodes like rising CO2 levels, erratic rainfall pattern, high temperature and moisture stress leaving deleterious imprints in physiology, disease resistance, fertility and productivity. Globally climate change is anticipated to pull down groundnut productivity by 11-25%. Agronomic manipulations like altered time of sowing, intercropping and irrigation management helps in microclimate modification towards reaping higher productivity and economic returns in groundnut. Methods: Field experiments were conducted during 2019-2021 on sandy clay loam soil (Fluventic Ustropept) in a Randomized Block Design with three factors viz., differential cropping systems (sole groundnut, groundnut + red gram intercropping), rainfed and irrigation systems and differential sowing windows (Second fortnight of June, first and second fortnights of July). Growth parameters, physiological traits viz., photosynthetic rate, transpiration rate, stomatal conductance, canopy temperature and light interception, incidence of foliar diseases viz., leaf spot and rust; soil borne disease viz., stem rot, root rot and productivity of groundnut were recorded at critical crop growth stages. Result: Canopy temperature was higher in sole groundnut system while light interception was higher in groundnut - redgram intercropping system, however system productivity did not register statistical superiority between the cropping systems. Irrigated system exerted its influence over rainfed system in terms of pod and kernel yield of groundnut. Sowing of groundnut during second fortnight of June was beneficial than July sowing in pod and kernel yield of groundnut due to uniform distribution of rainfall during the growth and reproductive phases of crop. Although differential cropping systems did not register their impact on disease incidence of groundnut, irrigated system and first sowing windows recorded minimum incidence of root rot, stem rot, early leaf spot, late leaf spot and rust diseases compared to rainfed system and July sowing.

Nitrogen management strategies on plant growth and severities of Sclerotinia stem rot of canola in eastern Canada

Effective nitrogen (N) management strategies are important for ensuring a balance between optimizing plant growth and minimizing disease damage. A field experiment was conducted for three years to (i) assess the effects of N fertilizer application on the growth and seed yield of canola, and severities of Sclerotinia stem rot (SSR), and (ii) determine a reasonable N-rate for optimizing plant growth and minimizing the loss from SSR in eastern Canada. The experiment was designed with factorial combinations of eight N treatments and two canola hybrids. All N-treatments reduced canola emergence with increasing preplant N application rates above 100 kg ha–1, but had a positive impact on plant height, fresh weight, dry weight and seed yield. The development of SSR showed differential responses to N application rates. Of all the treatments, the split application (50 kg N ha–1 at preplant plus 100 kg N ha–1 side-dressed at the 6-leaf stage) increased canola growth, and often produced the highest or similar seed yields to those of equivalent N rate applied as preplant. At the 150 kg ha–1 N rate, no severe development of SSR was observed in either preplant-only or split application. Overall, this study demonstrates that the split-N management strategy (50+100 kg ha–1) maintained a balance between enhancing plant growth and mitigating the negative impacts of SSR on canola.

Genetic Mapping and Genomic Prediction of Sclerotinia Stem Rot Resistance to Rapeseed/Canola (Brassica Napus L.) at Seedling Stage

The lack of complete host resistance and a complex resistance inheritance nature between rapeseed/canola and Sclerotinia sclerotiorum often limits the development of functional molecular markers that enable breeding for sclerotinia stem rot (SSR) resistance. However, genomics-assisted selection has the potential to accelerate the breeding for SSR resistance. Therefore, genome-wide association (GWA) mapping and genomic prediction (GP) was performed using a diverse panel of 337 rapeseed/canola genotypes. Three-weeks old seedlings were screened using the petiole inoculation technique (PIT). Days to wilt (DW) up to 2 weeks and lesion phenotypes (LP) at 3, 4, and 7 days post inoculation (dpi) were recorded. A strong correlation (r = -0.94) between DW and LP_4dpi implied that a single time point scoring at four days could be used as a proxy trait. GWA analyses using single-locus (SL) and multi-locus (ML) models identified a total of 35, and 219 significantly associated SNPs, respectively. Out of these, seventy-one SNPs were identified by a combination of the SL model and any of the ML models, at least two ML models, or two traits. These SNPs explained 1.4-13.3% of the phenotypic variance, and considered as significant, could be associated with SSR resistance. Eighty-one candidate genes with a function in disease resistance were associated with the significant SNPs. Six GP models resulted in moderate to high (0.45-0.68) predictive ability depending on SSR resistance traits. The resistant genotypes and significant SNPs will serve as valuable resources for future SSR resistance breeding. Our results also highlight the potential of genomic selection to improve rapeseed/canola breeding for SSR resistance.

First Report of Seedling Stem Rot on Jinxianlian (Anoectochilus roxburghii) Caused by Fusarium oxysporum in China

Anoectochilus roxburghii is an important Chinese herbal medicine plant belonging to Orchidaceae and known as Jinxianlian. This orchid is cultivated and mostly adopted to treat diabetes and hepatitis. About 2 billion artificially cultivated seedlings of Jinxianlian are required each year and approximately $600 million in fresh A. roxburghii seedlings is produced in China. From 2011, sporadic occurrence of stem rot on Jinxianlian have been observed in greenhouses in Jinhua City (N29°05′, E119°38′), Zhejiang Province. In 2018, nearly 30% of seedlings of Jinxianlian grown in greenhouse conditions were affected by stem rot in Jinhua City. Symptoms initially occurred in the stem at the soil line causing dark discoloration lesions, rotted tissues, wilting, and eventually leading to the death of the plants. A total of 23 diseased seedlings collected from seven different greenhouses were surface sterilized with 1.5% sodium hypochlorite for 3 min, then rinsed in water. Pieces of tissues disinfected from each sample were plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days (Kirk et al. 2008). A total of 19 isolates were recovered. They developed colonies with purple mycelia and beige or orange colors after 7 days of incubation under 25°C on PDA and carnation leaf agar (CLA) media (Kirk et al. 2008; Zhang et al. 2016). Colonies on PDA had an average radial growth rate of 3.1 to 4.0 mm /d at 25°C. Colony surface was pale vinaceous, floccose with abundant aerial mycelium. On CLA, aerial mycelium was sparse with abundant bright orange sporodochia forming on the carnation leaves. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.7 to 9.3 × 1.3 to 2.9 μm) (n=19). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (27.4 to 35.6 × 3.2 to 4.2 μm) (n=19). These morphological features were consistent with Fusarium oxysporum (Sun et al. 2008; Lombard et al., 2019). To confirm the identification based on these morphological features, the internal transcribed spacer region (ITS) and translation elongation factor1 (TEF) were amplified from the DNA of 3 out of 19 isolates chosen at random respectively using the set primer ITS1/ITS4 and EF1/ EF2 (Sun, S., et al. 2018; Lombard et al., 2019). BLAST analysis revealed that the ITS sequences (OK147619, OK147620, OK147621) had 99% identity to that of F. oxysporum isolate JJF2 (GenBank MN626452) and TEF sequence (OK155999, OK156000, OK156001) had 100% identity to that of F. oxysporum isolate gss100 (GenBank MH341210). A multilocus phylogenetic analysis by Bayesian inference (BI) and maximum likelihood (ML) trees based on ITS and TEF indicated that the pathogen grouped consistently with F. oxysporum. Three out of 19 isolates chosen at random were selected to evaluate pathogenicity. Uninfected healthy A. roxburghii seedlings about 40 day-old planted in sterilized substrates were sprayed with distilled water containing 2 x 106 conidia per ml suspensions as inoculums, and plants sprayed with distilled water alone served as controls. Plants were then incubated at 25°C and 85% relative humidity. Ten plants were inoculated for each isolate. After 10 days, all plants inoculated developed stem rot symptoms, while control plants remained healthy. Cultures of Fusarium spp. were re-isolated only from inoculated plants with the frequency of 100% and re-identified by morphological characteristics as F. oxysporum, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of F. oxysporum causing stem rot on A. roxburghii seedlings. As F. oxysporum is a devastating pathogenic fungus with a broad host range, measures should be taken in advance to manage stem rot of A. roxburghii.

First Report of Fusarium falciforme (FSSC 3+4) Causing Rot of Industrial Hemp (Cannabis sativa) in California

Industrial hemp (Cannabis sativa) is a newly legal crop in California that is grown for cannabidiol oil, fiber and seed. In August 2019, whole plant decline and root rot were observed affecting <5% of plants in two industrial fields in Fresno County, CA. Symptoms included chlorotic, collapsed foliage, stem vascular discoloration, and root rot with abundant mycelial growth. Stem and root segments (1-2 cm) from three to five diseased plants were agitated in 0.1% tween-20 and soaked in 70% ethanol for 30 s and 1% NaOCl for 2 min. After incubating for 5 to 7 days on 1:10 potato dextrose agar (PDA) amended with tetracycline, Fusarium selective medium (FSM), and PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) medium, white to pale cream aerial mycelium emerged from tissue of all plants on PDA and FSM but not PARP. Isolates cultured on 0.1% potassium chloride agar formed heads of microconidia on long monophialides consistent with the Fusarium solani species complex (FSSC) (Leslie and Summerell 2008). To obtain pure cultures of two isolates (CS529 and CS530), a single-hyphal tip was excised and grown on PDA. DNA was extracted from actively growing mycelium (PrepMan Ultra kit). The translation elongation factor gene (EF-1α) was amplified via PCR using EF1/EF2 primers (O’Donnell et al. 1998). Sequences of the two isolates were identical and deposited under accession number MW892973 in GenBank. The 599 bp sequence was 99.33% identical to FSSC 3 + 4 (Fusarium falciforme) accessions FD_01443_EF-1a based on FUSARIUM-ID BLAST analysis. To evaluate pathogenicity, stems of hemp plants (cv. ‘Berry Blossom’; n=8 plants per isolate) were wounded by penetrating the epidermis in an area about 0.5-cm square by 1-mm deep and 8-inches above the soil line. A 0.5 cm-diameter plug of 7-day old F. falciforme-colonized PDA was placed against the wound. Inoculation sites were loosely wrapped with parafilm for 2 days. A negative control consisted of a sterile PDA plug (n=3). Treatments were arranged in a completely randomized design in a greenhouse. The experiment was conducted once, due to regulatory restrictions at campus facilities. At 61 days post-inoculation, external stem lesions were significantly larger in diameter (P < 0.05; Tukey’s HSD) in plants inoculated with CS529 (8 ± 1 mm) compared to the control (2 ± 0 mm), and larger but not significant for CS530 (6 ± 1 mm). Internal stem lesions (i.e., rot in stele) were observed in plants inoculated with CS529 (9 ± 3 mm); stem rot was very minor in plants treated with CS530 (1 ± 1 mm) and nonexistent for control plants. No other disease symptoms were observed. F. falciforme was isolated from stems of CS529- and C530-inoculated plants. Sequences of re-isolates matched 100% with accession MW892973. These results suggest that F. falciforme causes rot in hemp in California. These studies specifically confirm stem rot abilities; field observations of root rot indicate root rotting abilities, but further tests are needed for confirmation. This is the first report of F. falciforme causing disease in industrial hemp. FSSC was described as causing foot rot in hemp in Italy (Sorrentino et al. 2019), but these isolates belonged to phylogenetic species 5 (F. solani) not F. falciforme. In addition, F. falciforme was reported as causing root rot in hydroponically grown cannabis (Punja and Rodriguez 2018). These studies provide the foundation for development of management tools for hemp disease.