تغني البيورينات بكتيريا الزائفة المرتبطة بالجذور وتحسن نمو فول الصويا البري تحت ضغط الملح

النتائج

تأثر نمو فول الصويا البري بضغط الملح

يؤدي ضغط الملح إلى استجابات مميزة في تنوع البكتيريا والأنواع السائدة بين الأقسام

لفحص كيفية تأثير إجهاد الملح على استقطاب البكتيريا، قمنا بمراقبة تركيب المجتمع البكتيري في الجذر، وتربة الجذور، والتربة العامة باستخدام تسلسل Illumina MiSeq لمنطقة V5-V7 من جين 16S rRNA عبر ثلاث نقاط زمنية (1، 7، و14 يومًا بعد إجهاد الملح). أظهرت المقارنة بين المعالجة الضابطة ومعالجة الملح عدم وجود اختلافات ملحوظة في تنوع شانون داخل الجذر في بداية إجهاد الملح (اليوم 1)، بينما أظهرت جميع معالجات الملح (باستثناء 300 مللي مول).

لتحديد تأثير الملح على الميكروبات المرتبطة بالجذور، قمنا بتحليل ملفات التركيب على مستوى الشعبة والترتيب للعينات الضابطة والعينات المعالجة بالملح. بعد 1-2 أسبوع (اليوم 7 واليوم 14) من

يزداد وفرة البكتيريا الزائفة بشكل كبير في الجذور المعالجة بالملح وتربة الجذور.

من بين أعلى 10 أجناس تغيرت، أظهرت بكتيريا الزائفة (المغذية في العلاجات المالحة) أعلى وفرة (الوفرة النسبية التراكمية) ) في كل من عينات الجذر والريزوسفير (الشكل 3 أ، ب)، في حين أن جميع الأجناس التي تغيرت في التربة الكثيفة وُجدت بوفرة منخفضة (جميع ) (الشكل 3ج). تم إثراء البكتيريا الزائفة في جميع الجذور المعالجة بالملح، وزادت وفرتها النسبية من في مجموعة التحكم إلى ، و في معالجات 100 و 200 و 300 مليمول من NaCl، على التوالي (الشكل 3أ). داخل مناطق الجذور، كانت بكتيريا Pseudomonas أيضًا غنية بشكل كبير في معالجات الملح بزيادة تصل إلى 16 ضعفًا مقارنةً بمجموعة التحكم (الشكل 3ب، د). تتماشى هذه النتائج مع نتائج تحليل جين 16S rRNA، حيث أظهرت التحليلات المستندة إلى الحمض النووي والحمض النووي الريبي للميكروبات في منطقة الجذور

عزلتان من بكتيريا الزائفة أنقذتا نمو فول الصويا البري تحت ضغط الملح

تم زراعة بذور فول الصويا البري لمدة 14 يومًا في تربة معقمة، تلتها فترة إجهاد ملحي لمدة 10 أيام. تم تلقيح السلالات XN05-1 و YE17 في التربة قبل إجهاد الملح. تم قياس معايير نمو الجذور والسوق لفول الصويا البري. لاحظنا أنه في ظل ظروف عدم وجود إجهاد ملحي، زادت فقط السلالة YE17 من نمو الجذور ولكن ليس من نمو السوق (الشكل 4). ومع ذلك، تحت إجهاد الملح، حسنت عزلات بكتيريا Pseudomonas XN05-1 و YE17 بشكل كبير من نمو الجذور والسوق (بما في ذلك الطول والوزن الطازج) لفول الصويا البري (الشكل 4). وهذا يشير إلى أن تعزيز نمو النبات الذي تسببه هذه السلالات كان محددًا بشكل أساسي لظروف الإجهاد الملحي. لتأكيد أن عزلات Pseudomonas استعمرت جذور فول الصويا البري، قمنا بتلقيح هاتين السلالتين في تربة معقمة بكميات متساوية لتحديد مستويات استعمار Pseudomonas تحت ظروف الملح وغير الملح باستخدام تقنية qPCR مع بادئات محددة. أظهرت النتائج أن القيمة المطلقة

زادت جينات وحمض نووي خلوية الحركة في منطقة الجذور المعالجة بالملح

مكون إفراز الجذور زانثين أعاد إنتاج إثراء بكتيريا الزائفة

دور الجين المرتبط بالحركة تشيو في الكيمياء الجذبية نحو البيورين وتحمل ملح النباتات

نقاش

هنا نقوم بتوصيف التغيرات في الميكروبيوم الجذري الناتجة عن الملح في فول الصويا البري. تحت ضغط الملح، حددنا العديد من الميكروبات الغنية والمستنفدة، وأظهرنا أن الملح أدى إلى زيادة ميكروبية أقوى نسبيًا في الجذر مقارنةً مع مناطق الجذور والتربة الكلية، مع زيادة تفضيلية عالية لبسودوموناس، والتي ثبت أنها تعزز نمو فول الصويا البري في هذه الدراسة. أخيرًا، تم توضيح التفاعل بين الإفرازات الجذرية والميكروبيوتا المرتبطة بالجذر المتغيرة تحت ضغط الملح.

الشكل 7 | رسم تخطيطي يوضح استقطاب البكتيريا الزائفة بواسطة فول الصويا البري تحت ضغط الملح. تم جذب بكتيريا الزائفة في التربة بواسطة الزانثين الذي تفرزه النباتات المتعرضة للضغط، وانتقلت نحو منطقة الجذور من خلال الحركة الكيميائية.

طرق

تصميم التجربة

أخذ العينات

قياس خصائص التربة

استخراج الحمض النووي، تسلسل جين 16S rRNA ومعالجة البيانات

تسلسل الميتاجينوم

استخراج RNA وتسلسل الميتا ترانسكريبتوم

جمع إفرازات الجذور وتسلسل الميتابولوم

qPCR للبكتيريا الكلية وPseudomonas

عزل البكتيريا وتجربة الدفيئة

استعمار Pseudomonas

تسلسل الجينوم

بناء طفرات cheW من Pseudomonas

اختبارات كيميائية كمية

تطبيق الزانثين الخارجي

التحليلات الإحصائية

ملخص التقرير

توفر البيانات

توفر الشيفرة

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شكر وتقدير

مساهمات المؤلفين

المصالح المتنافسة

معلومات إضافية

Purines enrich root-associated Pseudomonas and improve wild soybean growth under salt stress

Results

Wild soybean growth is affected by salt stress

Salt stress induces distinct responses in bacterial diversity and dominant taxa among compartments

To examine how salt stress affects bacterial recruitment, we monitored the bacterial community composition in the root, rhizosphere soil, and bulk soil using Illumina MiSeq sequencing of the V5-V7 region of the 16 S rRNA gene across three time points ( 1,7 , and 14 days after salt stress). Comparison of the control and salt treatments revealed no discernible differences in Shannon’s diversity within the root at early salt stress (Day 1), whereas all salt treatments (except for the 300 mM

To determine the effect of salt on root-associated microbiota, we analyzed the phylum- and order-level compositional profiles of the control and salt-treated samples. After 1-2 weeks (Day 7 and Day 14) of

Pseudomonas abundance increases dramatically in salt-treated roots and rhizosphere soils

Among the top 10 most changed genera, Pseudomonas (enriched in salt treatments) showed the highest abundance (cumulative relative abundance ) in both the root and rhizosphere samples (Fig. 3a, b), whereas all genera changed in the bulk soil were found in low abundance (all ) (Fig. 3c). Pseudomonas was enriched in all three salt-treated roots, and its relative abundance increased from in the control group to , and in the 100 , 200 , and 300 mM NaCl treatments, respectively (Fig. 3a). Within the rhizospheres, Pseudomonas was also highly enriched in salt treatments by up to 16 -folds than that in the control group (Fig. 3b, d). Consistent with the results of the 16S rRNA gene amplicon analysis, metagenomic DNA- and RNA-based analyses of the rhizosphere microbiota showed

Two Pseudomonas isolates rescued wild soybean growth under salt stress

Wild soybean seeds were grown for 14 days in sterile soil, followed by a 10-day period of salt stress. Strains XN05-1 and YE17 were inoculated into the soil before salt stress. The root and shoot growth parameters of wild soybean were measured. We observed that under nonsalt stress condition, only strain YE17 increased root growth but not shoot growth (Fig. 4). However, under salt stress, Pseudomonas isolates XN05-1 and YE17 significantly improved root and shoot growths (including length and fresh weight) of wild soybean (Fig. 4). This indicates that the plant growth enhancement induced by these strains was primarily specific to salt stress condition. To confirm that Pseudomonas isolates colonized the roots of wild soybean, we inoculated these two strains in sterile soil in equal amounts to quantify the levels of Pseudomonas colonization under salt and non-salt conditions using qPCR with specific primers. The results showed that the absolute

Cell motility genes and transcripts increased in salt-treated rhizosphere

Root exudate component xanthine reproduced Pseudomonas enrichment

The role of motility related gene chew in the chemotaxis toward purine and plant salt tolerance

Discussion

Here we characterize the salt induced root microbiome shifts in wild soybean. Under salt stress, we identified many enriched and depleted microbes, and demonstrated that salt led to relatively stronger microbial enrichment in the root compared with rhizosphere and bulk soil compartments, with highly preferential enrichment for Pseudomonas, which was evidenced to promote wild soybean growth in this study. Finally, the interaction between root exudates and altered rootassociated microbiota under salt stress were elucidated.

Fig. 7 | Schematic drawing illustrating the recruitment of Pseudomonas by wild soybean under salt stress. Soil Pseudomonas was attracted by xanthine secreted by stressed plant, and moved toward rhizosphere through chemotactic motility.

Methods

Experiment design

Sampling

Measurement of the soil properties

DNA extraction, 16 S rRNA gene amplicon sequencing and data processing

Metagenomic sequencing

RNA extraction and metatranscriptomic sequencing

Root exudates collection and metabolomic sequencing

qPCR for total bacteria and Pseudomonas

Bacterial isolation and greenhouse experiment

Pseudomonas colonization

Genome sequencing

cheW mutants construction of Pseudomonas

Quantitative chemotaxis assays

Exogenous xanthine application

Statistical analyses

Reporting summary

Data availability

Code availability

References

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