Evaluating Fungicide Timing, Efficacy, and Sensitivity as Well as Candidate Resistance Genes Against Fungi Causing Phomopsis Stem Canker in Sunflower

Phomopsis stem canker is one of the major diseases of sunflower (Helianthus annuus L.) caused by complex of Diaporthe spp., in the U.S. This disease became of economic importance after 2010 epidemic in Minnesota, North Dakota, and South Dakota and since then the prevalence of this diseases has increased due to increased average precipitation in these major sunflower growing regions of the U.S. The two important disease management strategies involve the use of fungicides and partially resistant hybrids. However, over the years, researchers and growers have seen varied results on Phomopsis stem canker disease severity reduction and yield gains, when it comes to the application of foliar fungicides. Moreover, information on fungicide sensitivity of the two predominant pathogens causing Phomopsis stem canker (D. gulyae and D. helianthi) is lacking. Additionally, information on the resistance mechanism and the genes conferring resistance to Diaporthe species could aid future marker-assisted breeding studies. Therefore, the study was planned with the following objectives to (1) assess the potential of fungicide active ingredients and application timings in managing Phomopsis stem canker; (2) determine the fungicide sensitivity of D. gulyae and D. helianthi against fluxapyroxad, pyraclostrobin, and tebuconazole fungicides; and (3) explore the sunflower response to infection by D. helianthi using RNA-sequencing. For the first objective, we analyzed foliar fungicide efficacy trials conducted between 2009 to 2021 in Minnesota, Nebraska, North Dakota, and South Dakota. The trials served as datasets to evaluate the best fungicide application timing and active ingredient combination for managing Phomopsis stem canker and preventing yield losses. A metaanalytical approach was used to analyze 56 location-years trials using non-linear regression to determine the effect of a total of seven application timings (single and sequential) and seven active ingredients (single and combination) on the disease severity index (DSI) and yield in comparison to the non-treated control (NTC) by calculating two effect sizes (Cohen’s f or Hedges’s g) corresponding to the differences in DSI and yield between the treated and NTC. The pooled effect was significant for both fungicide application timings [DSI (f=-0.43), yield (f=0.26)] and active ingredients [DSI (f=-0.51); yield (f=0.39)] on DSI and yield (P \u3c 0.0001). Based on the 95% confidence intervals (CI) around g, fungicide applications made at R1 growth stage (miniature floral head development) did not contain zero indicating a significant disease reduction (g=-0.57), and yield gain (g=0.33), in comparison to NTC. In addition, a split application of pyraclostrobin at R1 + R5 [DSI (g=-0.55)] outperformed NTC in managing the disease. Similarly, based on the 95% CI, the active ingredients fluxapyroxad + pyraclostrobin [DSI (g=-0.55), yield (g=0.36)], mefentrifluconazole + pyraclostrobin + fluxapyroxad [DSI (g=-2.23), yield (g=0.85)], and pyraclostrobin [DSI (g=-0.65), yield (g=0.43)] were able to significantly reduce the disease and prevent yield losses. This study suggests that use of pyraclostrobin based fungicides at R1 can reduce Phomopsis stem canker severity and increase yield when initial disease symptoms start to appear in the field. The second objective involved a proactive fungicide sensitivity monitoring program to determine the sensitivity of D. gulyae and D. helianthi to fluxapyroxad, pyraclostrobin, and tebuconazole fungicides. The assay was conducted under lab conditions (22±2⁰C in dark) using a total of 103 isolates collected from Minnesota, Nebraska, North Dakota, and South Dakota. In addition, three baseline isolates [one isolate of D. gulyae (ex-type BRIP 54025) from Australia and two D. helianthi isolates (ATCC 201540 and 52763), obtained from ATCC collection] were used in the study. Technical grade products of the three fungicides were dissolved in acetone to prepare stock solutions. Following which, water-agar media was serially amended with different concentrations of fluxapyroxad, pyraclostrobin + SHAM (salicylhydroxamic acid – 20 μg ml-1), and tebuconazole, using poison food technique. A 0.6-cm inverted mycelial plug of D. gulyae or D. helianthi was placed onto the center of each fungicide amended Petri-dish and a fungicide concentration of 0 μg ml-1 or acetone served as the control. For each fungus and fungicide combination, the experiment was arranged in a completely randomized design, with four replications per fungicide concentration and was repeated once. The plates were then incubated in dark at 22±2°C for 5 days (D. gulyae) and 7 days (D. helianthi), after which the radial growth of the fungus was measured at right angles using a scale. The percent mycelial growth inhibition was used to calculate the effective concentration that inhibited the growth of fungus by half (EC50) through non-linear regression. A significant effect of EC50 for the three fungicides on all isolates (P \u3c 0.0001) was observed using ANOVA Type Statistics (ATS). For D. gulyae, the mean EC50 values were 6.234, 0.919, and 0.245 μg ml-1 for fluxapyroxad, pyraclostrobin, and tebuconazole, respectively where six, 22, and 21 isolates for each fungicide had significantly greater EC50 (P \u3c 0.0001) than the baseline. While the mean EC50 values for D. helianthi, were 5.999, 0.171, and 0.127 μg ml-1 for fluxapyroxad, pyraclostrobin, and tebuconazole, respectively where three, three, and 13 isolates for each fungicide had significantly greater EC50 (P \u3c 0.0001) than the two baselines. The results indicate a possible decline in the sensitivity of Phomopsis species to fluxapyroxad, pyraclostrobin, and tebuconazole fungicides. This study establishes the first fungicide sensitivity monitoring of D. gulyae and D. helianthi against fluxapyroxad, pyraclostrobin, and tebuconazole fungicides program and the generated information from this study will be critical for future sensitivity assays. The last objective was to investigate the host pathogen interaction between D. helianthi isolates and sunflower genotypes using transcriptome analysis. For this, we carried out RNAseq analysis of one susceptible (PI 552934) and two partially resistant (PI 561918 and PI 509062) accessions at 0 and 72 hrs. post-inoculation (hpi) on infection of three D. helianthi isolates (DIA-59, DIA-130, DIA-197) to spot the sunflower transcriptome response and figure out the mechanisms underlying Phomopsis stem canker causing fungal disease resistance. The results showed a total of 249, 277, and 264 differentially expressed genes (P ≤ 0.001; log2FC \u3e2 o

Insect and Pest Management for Sustaining Crop Production Under Changing Climatic Patterns of Drylands

Climate change is alarming, particularly for agriculturists as it severely impacts the development, distribution, and survival of insects and pests, affecting crop production globally. Over time, climate change is drastically tumbling the crop productivity in all the cropping systems, whereas the dryland agriculture with existing low productivity is immensely hit. While all the existing species in drylands, including humans, are coping with extreme climate variations for millennia, future climate change predictions put dryland agriculture in a threat zone. Drylands support 38% of the world’s population; therefore, climate change coupled with population growth and global food security draws the attention of scientists towards sustainable crop production under changing trends. The intermingling and intermixing of various biological, hydrological, and geographical systems plus the anthropogenic factors continuously amplify the changes in the dryland systems. All of this brings us to one challenge: developing pest management strategies suitable for changing climatic patterns. In this complex agrology framework, integrated pest management (IPM) strategies, especially those involving early monitoring of pests using prediction models, are a way to save the show. In this chapter, we will summarize the direct and indirect effects of climate change on crop production, the biology of insect pests, the changing pest scenarios, the efficacy of current pest management tactics, and the development of next-generation crop protection products. Finally, we will provide a perspective on the integration of best agronomic practices and crop protection measures to achieve the goal of sustainable crop production under changing climatic trends of drylands

Seasonal variation of antioxidant enzymatic responses in the desiccation-tolerant bryophyte Syntrichia ruralis (Hedw.) Web.& Mohr.

Bryophytes are poikilohydric organisms that can be used as model plants to study desiccation tolerance mechanisms. The main objective of this study was to examine the activities of the antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT) and guaiacol peroxidase (POD) in the rehydrated and desiccated states in Syntrichia ruralis (Hedw.) Web. & Mohr. from two slopes, one North-east (NE) and one South-west (SW) facing and collected in different seasons. Our results showed seasonal variation in the enzymatic activities of APX, CAT and POD between the slopes in both the rehydrated and desiccated states. The mean value of all the activities of APX, CAT and POD and MDA contents (a measure of lipid peroxidation) tended to be higher in moss cushions collected from the NE compared to the SW facing slopes except in summer season. The mean values of all enzymatic activities were higher in desiccated states as compared with rehydrated states. Protein content has lower values in summer and winter season. Differences in the antioxidant activities of the mosses growing on the two slopes may reflect adaptations to desiccation stress

Mycobacterial Dormancy Regulon Protein Rv2623 as a Novel Biomarker for the Diagnosis of Latent and Active Tuberculous Meningitis

The present study was designed to investigate Rv2623 antigen, a major dormancy regulon protein of Mycobacterium tuberculosis (MTB) in CSF of suspected latent and active tuberculous meningitis (TBM) patients. A total of 100 CSF samples from TBM (n=31), suspected latent TBM (n=22), and suitable noninfectious control subjects (n=47) were collected and evaluated for Rv2623 antigen level using ELISA protocol. A significantly high (P<0.05) mean absorbance was observed in samples of suspected latent TBM and active TBM patients as compared to non-TBM control patients. However, no significant difference in Rv2623 level was observed between suspected latent TBM and TBM patients. Our preliminary findings suggest that Rv2623 may be useful as a potential biomarker for the diagnosis of the latent as well as active TBM infection. Futher evaluation of this biomarker in large number of samples is therefore needed to confirm the result

Impact of socioeconomic status and living condition on latent tuberculosis diagnosis among the tribal population of Melghat: A cohort study

Aims: To study socioeconomic status (SES) and living conditions (LC) as risk factors for latent tuberculosis infection (LTBI) and their impact on QuantiFERON-TB gold (QFT-G) and tuberculin skin test (TST) outcome for determining a better diagnostic test for LTBI in the malnourished tribal population of Melghat. Settings and Design: Six hundred sixty nine participants matching the inclusion criteria were recruited from 10 tribal villages of Melghat region, India. Subjects and Methods: Complete information related to various risk factors and test outcome was obtained on 398 participants, which was analyzed as per predefined conceptual framework. Factors were classified based on their relevance either at individual or household level, and subsequently based on the possibility of intervention. Data were partitioned into concordant and discordant sets depending on test agreement. Results: In concordant set, the two tests revealed that LTBI was significantly associated with smoking (adjusted odds ratio [aOR]: 2.64 [95% confidence interval [CI]: 1.03-6.79]), tobacco usage (aOR: 2.74 [95% CI: 1.50-4.99]), and malnourishment (aOR: 1.97 [95% CI: 1.12-3.48]) after basic adjustment. Inclusion of latent variable SES and LC in the model has mediating effect on the association of above factors with LTBI. Further, the association of SES and LC with LTBI in concordant set was unaltered in presence of other cofactors. From discordant set, results of QFT-G corroborated with that of concordant set. Conclusions: Poor SES and LC can be considered as strong risk factors linked with LTBI as compared to malnourishment, which is often targeted in such communities. Further, our study showed QFT-G test as a reliable tool in screening of LTBI in the tribal population of Melghat, India

Latent TB infection diagnosis in population exposed to TB subjects in close and poor ventilated high TB endemic zone in India.

The present study was designed to investigate the utility of Quantiferon TB gold (QFT-G) and Tuberculin skin test (TST) for diagnosis of latent TB infection (LTBI) in high crowding TB endemic zone of Nagpur, India and their comparison with associated risk factors.Out of 342 eligible participants, QFT-G and TST were performed in 162 participants.The prevalence of LTBI observed according to QFT-G and TST was 48% and 42% respectively, with an agreement of 52.47%. QFT-G positivity was associated with age while TST positivity was associated with body mass index (BMI). Duration of exposure emerged as a key risk factor significantly associated with both the tests.The prevalence of LTBI was quite high in the studied zone as detected by both the evaluated tests and thus, the combination of both the tests will be best predictive for LTBI in such high TB endemic regions

Laboratory Investigations on the Diagnosis of Tuberculosis in the Malnourished Tribal Population of Melghat, India

<div><p>Background</p><p>Malnutrition is a major risk factor for the development of tuberculosis (TB). In India, Melghat is among the tribal regions which consist of highest number of malnutrition cases. Because of the paucity of TB data from these malnourished areas there is an urgent need for the development and evaluation of improved TB diagnostic tests. In the present study, three in house developed diagnostic tests namely TB-Ag(antigen) ELISA, Adenosine deaminase (ADA) estimation and IS<i>6110</i> polymerase chain reaction (PCR) assay were investigated for the detection of <i>Mycobacterium tuberculosis</i> (<i>M. tb.</i>) infection.</p> <p>Methods</p><p>For investigation, blood samples were collected from 128 study subjects from six villages of Melghat tribal area and evaluated using three in house developed assays, namely TB-Ag ELISA, ADA estimation and IS<i>6110</i> PCR.</p> <p>Results</p><p>The TB-Ag ELISA method yielded 83% sensitivity and 94% specificity. The ADA and PCR assay gave a sensitivity of 61% and 49% and specificity of 62% and 98% respectively. A considerable good agreement of 82.81% (k=0.472) between TB-Ag ELISA and PCR was observed. The overall sensitivity of TB-Ag ELISA was significantly higher (p<0.05) than the ADA and PCR while PCR yielded highest specificity among all the three evaluated tests.</p> <p>Conclusions</p><p>We concluded that the routine use of TB-Ag ELISA can be useful for screening of suspected TB patients in the malnourished population where sophisticated laboratory set up is difficult.</p> </div

General characteristics of TB exposed cases (<i>n</i> = 162).

†<p>Out of 117.</p